Leica TCS SP5 User Manual
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Leica TCS SP5 User Manual

Leica TCS SP5 User Manual

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  • Page 1 Leica TCS SP5 User Manual...
  • Page 2 Published by: Leica Microsystems CMS GmbH Am Friedensplatz 3 D-68165 Mannheim (Germany) http://www.leica-microsystems.com http://www.confocal-microscopy.com Responsible for contents: Leica Microsystems CMS GmbH Copyright © Leica Microsystems CMS GmbH. All rights reserved.
  • Page 3 Notes about this manual..................7 Copyright ......................7 Patents ....................... 8 General ......................... 9 About these operating instructions ..............9 Intended use ....................... 9 TCS SP5 specifications ..................11 System overview ....................11 Dimensions ....................... 12 3.2.1 System with inverted stand:................12 3.2.2 System with upright stand................
  • Page 4 Overview of usable VIS/UV lasers..............36 5.11 Overview of usable MP lasers (IR lasers)............37 5.12 Safety label on TCS SP5 system ..............37 5.12.1 Inverted stand DMI 6000 CS ................38 5.12.2 Upright stand DM 5000/6000 CS ..............40 5.12.3...
  • Page 5 8.3.1 General structure of the user interface ............60 Key combinations ..................... 61 Introduction to Confocal Work................. 63 Preparation ....................... 63 9.1.1 The objective ....................63 9.1.2 Conventional microscopy................64 9.1.3 Why scan? ...................... 67 9.1.4 How is an optical section created? ..............68 Acquiring optical sections .................
  • Page 6 9.5.3 Lines ....................... 93 9.5.4 Planes......................93 9.5.5 Spaces (time-space)..................93 9.5.6 FRAP measurements ..................94 Spectral series ....................94 9.6.1 Data acquisition and utilization ............... 94 9.6.2 About spectral resolution ................94 Combinatorial analysis ..................95 Switching off the system .................. 97 Disposal ......................

  • Page 7: Notes About This Manual

    The information in this manual is subject to modification at any time and without notification. All rights to this documentation are held by Leica Microsystems CMS GmbH. Adaptation, translation and reproduction of text or illustrations (in whole or in part) by print, photocopy, microfilm or other method (including electronic systems) is not allowed without express written permission from Leica Microsystems CMS GmbH.

  • Page 8: Patents

    Patents The product is protected under the following US patents: 5,886,784; 5,903,688; 6,137,627; 6,222,961; 6,285,019; 6,311,574; 6,355,919; 6,423,960; 6,433,814; 6,444,971; 6,466,381; 6,510,001; 6,614,526; 6,654,165; 6,657,187; 6,678,443; 6,687,035; 6,738,190; 6,754,003; 6,801,359; 6,831,780; 6,850,358; 6,867,899. Further patents are pending.

  • Page 9: General

    Suite Advanced Fluorescence (LAS AF). The Leica TCS SP5 is supplied with the latest version of the licensed Leica Application Suite Advanced Fluorescence. To maintain information on the most current level, the description of software functions was intentionally omitted from these operating instructions. Instead, you...

  • Page 11: Tcs Sp5 Specifications

    3. TCS SP5 specifications System overview Figure 1: System components (overview) TCS SP5 Scanner Main switch board TCS workstation Supply unit Control panel Microscope stand...

  • Page 12: Dimensions

    3.2.1 System with inverted stand: 1400 mm 730 mm 600 mm 2540 mm 1200 mm Figure 2: Dimensions of the TCS SP5 with inverted stand 3.2.2 System with upright stand 1490 mm 730 mm 600 mm 2400 mm 1200 mm...

  • Page 13: Requirements Regards Ambient Conditions

    Installing the system in darkened rooms is also advisable. For installation, maintenance and transport, the TCS SP5 system requires doors with inside spans of 1.00 m. With regard to the load-bearing capacity of the floor, please note that the system will apply a static load of 200 kg/m².

  • Page 14: Electrical Connections

    Waste heat/required cooling performance The TCS SP5 system features a maximum power consumption of 3.2 kW (VIS system) or 6.2 kW (MP system), respectively. For the specifications of external lasers such as UV and MP lasers, please refer to the manufacturer's documentation.

  • Page 15: Safety Instructions And Their Meaning

    4. Safety instructions and their meaning DANGER This kind of warning alerts you of an operating procedure, practice, condition, or statement in the operating instructions that must be strictly observed and followed, as otherwise you expose yourself to the risk of fatal injury. WARNING! LASER RADIATION A laser warning points out an operation, a process, a condition or an instruction that must be observed strictly to prevent serious eye injuries to the persons using...

  • Page 17: Safety Instructions

    5. Safety instructions The instrument is Class 3B or 4 laser equipment (depending on the laser used) in accordance with IEC/EN 60825-1. This laser equipment may be operated only by persons who have been instructed in the use of the system and the potential hazards of laser radiation. As it is not possible to anticipate every potential hazard, please take care and apply common sense to the installation, operation and maintenance of this product.

  • Page 18: Which Laser Class Does The Product Have

    For use in the USA: Laser Products U.S. Food and Drug Administration (FDA) (”Complies with FDA performance standards for laser products CDRH 21 CFR 1040.10: except for deviations pursuant to laser notice No. 50, dated 26 July, 2001. For the scope of the CDRH/FDA (USA), the designation Laser Class 3B in the text must be replaced by IIIb and Class 4 by IV.

  • Page 19: Laser Class For Mp Systems

    Deactivating or damaging these safety devices or any intervention in any of these safety devices may lead to serious eye injuries, physical injuries or property damages. In these cases, Leica Microsystems CMS GmbH does not assume any liability. According to IEC/EN 60825-1: "Safety of laser products, Part 1: Equipment classification, requirements and user's guide,"...

  • Page 20: Safety Notes For The User

    If repairs or service measures are performed that require opening parts of the housing, only trained Leica service technicians may occupy the room in which the laser scanning microscope is located. Leica Microsystems CMS GmbH will not be liable for damages resulting from non observance of the above information.

  • Page 21: Safety Notes For Operation

    Safety Notes for Operation Figure 4: Specimen area of upright and inverted stand During scanning, the laser radiation is freely accessible after exiting the objective in the specimen area of the laser scanning microscope. This circumstance demands special attention and caution. If the laser radiation comes in contact with the eyes, it may cause serious eye injuries.
  • Page 22 Never deactivate the laser protection devices. Please read the chapter "Laser protection devices" to familiarize yourself with the safety devices of the laser scanning microscope. Do not introduce any reflective objects into the laser beam path. Never change specimens during scanning. To change specimens proceed as follows: Upright microscope Inverted microscope...

  • Page 23: Specific Safety Notes

    Do not use an S70 microscope condenser. The large working distance and the low numeric aperture of the S70 microscope condensers could result in a threat from laser radiation. Therefore, only S1 and S28 Leica microscope condensers should be used.

  • Page 24: De-Energizing The System

    Contact with liquids or the entry of liquids into the housing must be avoided. 5.8.2 De-energizing the system The main circuit breaker is located on the right rear side of the supply unit. It is used to de- energize the complete system using a single switch.(Figure 5). The main circuit breaker functions as a switch and as an overcurrent fuse.

  • Page 25: Laser Safety Devices

    Figure 6: Power outlet strip, rear side of supply unit Laser safety devices The light of all employed VIS lasers (wavelength range 400 - 700nm, visible spectrum) and UV lasers (wavelength range < 400nm, invisible) is fed through an optical waveguide and, therefore, completely shielded until it leaves the microscope objective and reaches the specimen.

  • Page 26: Emissions Warning Indicators

    Figure 7: Detachable-key switch for the internal lasers For lasers that are not connected as described above, please refer to the supplied manual of the laser manufacturer for the position of the detachable-key switches. 5.9.3 Emissions warning indicators The operational readiness of lasers located in the supply unit is signaled by an emission warning indicator (Figure 8).

  • Page 27: Remote Interlock Connector On The Supply Unit

    Scanning of the specimen is not activated after being switched on properly (laser radiation in the specimen area). Contact Leica Service immediately. For lasers whose readiness is not indicated as described above, please refer to the supplied manual of the laser manufacturer for the location of the emission warning indicator.

  • Page 28: Remote Interlock Connectors On Additional External Lasers

    Figure 10: Example of a remote interlock Due to the live voltage of 100 – 240 V, replacing the shorting plug by an external interrupt circuit (e.g. door interlock switch) may only be performed by a qualified electrician. 5.9.5 Remote interlock connectors on additional external lasers For lasers whose remote interlock connector is not indicated in Chapter 5.9.4, please refer to the supplied manual of the laser manufacturer for the location of the remote interlock connector.

  • Page 29: Function And Position Of Safety Switches

    Figure 11: Position of the interlock connector 5.9.7 Function and position of safety switches When the safety switches are released, the light path of the laser beam is interrupted. Figure 12: Position of the transmitted-light illumination arm (1) and switching from scan mode to eyepiece (2).

  • Page 30: Transmitted-Light Lamp Housing For Inverted Stands

    Type of Position Activated by: Activated if: Function microscope The illuminator Prevents laser light Transmitted-light Inverted stand arm is tilted (e.g. while working on the illuminator arm DMI 6000 CS for working on the specimen. specimen). Motorized Prevents stray light if The deflection changeover the user switches...

  • Page 31: Transmitted-Light Lamp Housing For Upright Stands

    Figure 14: Connecting the transmitted light lamp housing If a lamp housing is not attached to the microscope stand and there is no cover, the laser may not be switched on, as otherwise laser radiation can be emitted. 5.9.9 Transmitted-light lamp housing for upright stands During the time when no transmitted-light lamp housing is connected to the upright microscope stand, the opening must be tightly covered with the cap provided with the system to prevent laser radiation from exiting (Figure 15).

  • Page 32: Mirror Housing On Upright Stand

    Remove the lamp housing. Perform the intended tasks at the lamp housing. After finishing the tasks, screw the new lamp housing back onto the microscope stand. Figure 16: Connecting the transmitted light lamp housing If neither a lamp nor mirror housing is attached to the microscope stand and there is no cover, the laser may not be switched on, as otherwise laser radiation can be emitted.
  • Page 33 Figure 17: Cover If your upright microscope stand is equipped with a mirror housing, note the following: • The interlock connector on the mirror housing (see Figure 18, arrow) must be connected to the scan head at all times. • The unused output on the mirror housing must be covered with the cover provided (see Figure 18, position 1).

  • Page 34: Special Laser Safety Equipment

    5.9.11 Special laser safety equipment 5.9.11.1 Safety beam guide and beam collector On inverted microscopes, the safety beam guide and the beam collector serve as protection against laser radiation emission and are located between the condenser base and the transmitted-light detector (see Figure 19). Safety beam guide Beam collector (illustrated is the version of the beam...

  • Page 35: Eye Protection For Mp Systems

    IR laser radiation are provided with the system when delivered. The supplied eye protection only provides safe protection against the infrared lasers supplied by Leica Microsystems CMS GmbH. At this point, it is our duty to underline the following requirement: When using MP systems with upright microscope stands every single person present in the room should wear safety goggles during scanning.

  • Page 36: Overview Of Usable Vis/Uv Lasers

    5.10 Overview of usable VIS/UV lasers The laser scanning microscope features a combination of the lasers listed below. Maximum Maximum luminous power luminous power Wavelength at laser output in focal plane Laser type [nm] Pulse duration [mW] [mW] Continuous wave Diode 405 <...

  • Page 37: Overview Of Usable Mp Lasers (Ir Lasers)

    Table 2 Table of usable MP lasers 5.12 Safety label on TCS SP5 system The corresponding safety labels are selected dependent on the laser configuration (VIS, UV, MP) and attached in the following locations either in the English or German language.

  • Page 38: Inverted Stand Dmi 6000 Cs

    5.12.1 Inverted stand DMI 6000 CS Angled rear view of right side of stand Figure 21: Safety label for DMI 6000 CS inverted stand...
  • Page 39 Angled rear view of right side of stand Figure 22: Safety label for DMI 6000 CS inverted stand...

  • Page 40: Upright Stand Dm 5000/6000 Cs

    5.12.2 Upright stand DM 5000/6000 CS Angled rear view of right side of stand: Figure 23: Safety label for DM 5000/6000 CS upright stand...
  • Page 41 Rear view of stand: Figure 24: Safety label for DM 5000/6000 CS upright stand...

  • Page 42: Scan Head

    5.12.3 Scan head Angled front view of left side of scan head: Figure 25: Safety label for the scanner...

  • Page 43: Supply Unit

    5.12.4 Supply unit View of TCS SP5 supply unit: Figure 26: Safety label for the supply unit TCS SP 5 (front side)

  • Page 44: Mp Beam Coupling Unit

    5.12.5 MP beam coupling unit Angled front view of the right side of the beam coupling unit MP: Figure 27: Safety label for the beam coupling unit MP (top side)

  • Page 45: Cover (For Replacement Flange)

    5.12.6 Cover (for replacement flange) View from front on the cover: Figure 28: Cover If the replacement flange for transmitted light is not equipped with a functional module such as a lamp housing, place a cover over the opening for laser safety reasons.

  • Page 46: Mirror Housing

    5.12.7 Mirror Housing View from front on the mirror housing: Figure 29: Safety label for mirror housing (top)

  • Page 47: Installation And Storage Site Requirements

    5.13 Installation and storage site requirements This device was designed for use in a lab and may not be set up in areas with medical devices serving as life-support systems such as intensive-care wards. This equipment is designed for connection to a grounded (earthed) outlet. The grounding type plug is an important safety feature.

  • Page 48: Scanner Cooling

    Inform Leica or a Leica-approved service facility immediately. The coolant contains an irritating substance. Avoid eye and skin contact. The scanner cooling system must be serviced by Leica or a Leica-approved service facility every two years.

  • Page 49: Care And Maintenance

    Persistent dirt can be removed from glass surfaces by means of pure alcohol or chloroform. If an objective lens is accidentally contaminated by unsuitable immersion oil or by the specimen, please contact your local Leica branch office. for advice on the use of certain solvents for cleaning purposes.

  • Page 50: Cleaning The Microscope Surface

    Never use acetone, xylene or nitro thinners as they attack the varnish. All LEICA components and systems are carefully manufactured using the latest production methods. If you encounter problems in spite of our efforts, do not try to fix the devices or the accessories yourself, but contact your Leica representative.

  • Page 51: Startup Of The System

    3. Typing your password identifies you as a valid user for this system. The default user name for the Leica TCS SP5 system is "TCS_User". A standard password was not set. It is recommended setting up a separate user ID for each user (set up by the system administrator).

  • Page 52: Setting Up Users

    AF software. For information about setting up users, please refer to the chapter "Setting Up Users" in this manual. After logging on with your user ID, you may change your password by pressing the keys Ctrl, Alt, and Del at the same time. Then click on Change password.

  • Page 53: Starting Up

    Starting up Turn on the control computer on the main switch board (see Figure 30, page 51). Check whether the microscope stand is switched on. If the readiness indicator (Figure 31, 1) on the electronics box for microscopes is lit, the stand is operating. If the readiness indicator is not lit, activate the toggle switch (Figure 31, 2) of the E-box.
  • Page 54 Figure 32: Turning on the scanner 5. Switch on the lasers on the main switch board. Figure 33: Switching on the laser The power supplies and ventilation of the system have been started. 6. Engage the detachable-key switch on the main switch board.

  • Page 55: Starting The Las Af

    Figure 34: Activating the detachable key switch Laser radiation may be present in the specimen area as of this time. Follow the safety instructions given in chapter 5. Please follow the instructions in the chapter on switching off the system to switch off the system.
  • Page 56 Figure 36: Resonant or non-resonant mode 3. Start the LAS AF by clicking on "OK". Figure 37: LAS AF start window You are now in the base view of the LAS AF. Figure 38: LAS AF base view...

  • Page 57: Introduction To Las Af

    8. Introduction to LAS AF General The LAS AF software is used to control all system functions and acts as the link to the individual hardware components. The experimental concept of the software allows for managing the logically interconnected data together. The experiment is displayed as a tree-structure in the software and features export functions to open individual images (JPEG, TIFF) or animations (AVI) in an external application.

  • Page 58: Calling Online Help

    8.2.2 Calling Online Help The online help can be called up in three ways: In the respective context (context-sensitive) Via the Help menu With the key combination CTRL + F1 In the respective context (context-sensitive) Click on the small question mark located in the top right corner of every dialog window. Online help opens directly to the description for the corresponding function.

  • Page 59: Full-Text Search With Logically Connected Search Terms

    8.2.3 Full-text search with logically connected search terms Click on the triangle to the right of the input field on the Search tab to view the available logical operators. Select the desired operator. Enter the second search term you would like to associate with the first search term behind the operator: Examples Results...

  • Page 60: Structure Of The User Interface

    Structure of the user interface 8.3.1 General structure of the user interface The user interface of the LAS AF is divided in five areas: Figure 39: LAS AF user interface Menu bar: The different menus for calling functions are available here Arrow symbols: Operating step with the individual functions.

  • Page 61: Key Combinations

    Tab area: Different tabs belong to every operating step (arrow symbol) in which the settings for the experiment can be made. Acquire Experiments: Directory tree of opened files Setup: Hardware settings for the current experiment Acquisition: Parameter settings for the image recording Process Experiments: Directory tree of opened files Tools: Directory tree with all the functions available in the respective...

  • Page 63: Introduction To Confocal Work

    9. Introduction to Confocal Work Preparation The following sections describe a number of basic procedures that cover most of the tasks related to the instrument. a) upright microscope 1 objective 2 coverslip 3 seal 4 glass slide 5 stage focus b) inverted microscope 1 embedding 2 specimen...

  • Page 64: Conventional Microscopy

    Medium Refractive Index Water 1,333 1,335 Glycerol 80 % (H 1,451 Vectashield 1,452 Glycerol 1,462 Moviol 1,463 Kaisers Glycerol Gel 1,469 Glass 1,517 1,518 Canada Balsam 1,523 Table 3 Immersion media When using immersion objectives, ensure that an adequate quantity of immersion medium is applied between the front lens of the objective and the specimen.
  • Page 65 Filter cubes (Figure 41) suitable to the fluorescence must be positioned in the beam when viewing the specimen via the eyepieces. For more information on selecting fluorescence filter cubes, please refer to the Leica fluorescence brochure or contact your Leica partner. For a selection of filter cubes, see Table 4 below.
  • Page 66 BP 527/30 BP 546/14 LP 590 N2,1 BP 515-560 LP 590 BP 546/12 BP 600/40 BP 545/30 BP 610/75 BP 620/60 BP 700/75 BP 500/20 BP 535/30 Table 4 Selection of filter cubes for Leica research microscopes and associated filter specifications.

  • Page 67: Why Scan

    9.1.3 Why scan? Specimens must be illuminated over the smallest possible area to achieve a true confocal image – this is essential to attaining truly thin optical sections. That has been achieved when the illumination spot is diffraction limited; i.e. it cannot be made physically smaller.

  • Page 68: How Is An Optical Section Created

    Figure 43: Smallest possible, diffraction-limited illumination spot (Airy disk). Below: an intensity profile. To scan a line, the mirror must travel across the field of view. The y mirror is then moved a small amount, after which the x mirror then scans the next line. The signals from the specimen are written to an image memory and can be displayed on the monitor.
  • Page 69 A value of 1 Airy is a very good compromise and is selected automatically by the Leica TCS SP5. A dialog is available to set smaller or larger diameters if required. Playing with this parameter to study its...

  • Page 70: Acquiring Optical Sections

    Figure 45: Use the "Acquire" arrow button to acquire data in all Leica LAS AF applications. The Leica TCS SP5 contains many functions in its user interface that reflect its wide range of potential applications. The functions not needed for a given application are disabled, however, to ensure efficiency and ease of use.
  • Page 71 Alternatively, a single image can be captured. This image is then stored in the experiment and can be retrieved later or stored on any data medium. Individual image capture has the advantage of only exposing the specimen once, but is less convenient if additional setup work is required.

  • Page 72: Illumination

    I (t) Figure 48: LiveDataMode supports the continuous acquisition of data while changing setting parameters, manipulating the specimen or performing bleaching sequences between the individual captures. The clock continues running throughout the experiment and intensity changes in interesting areas can be rendered graphically online. The setting parameters for a simple optical section are described and discussed below.

  • Page 73: Beam Splitting

    Figure 49: Selecting the illumination intensity (1) via acousto-optic tunable filter (AOTF, 2) and selection of the emission band in the SP detector (3). When selecting the "FITC" parameter set, the 488nm argon line and a suitable band between 490nm and 550nm is set. The entire beam path is represented graphically on the user interface.

  • Page 74: Emission Bands

    Figure 50: Excitation spectrum of a fluorescence stain (blue) and emission spectrum (red). An excitation in the maximum (Exc1) would result in only a narrow band to be collected on the emission side (Em1). A significantly broader emission band (Em2) is available from an excitation in the blue range, at which point the intensity of the laser can be increased without detrimental effects.
  • Page 75 Using the excitation lines and displayed emission characteristics for orientation and adapting the emission band using the Leica SP® detection system is thus very convenient. This is also possible during live acquisition of images. The effects of settings on the images are immediately apparent and suitable values can thus be selected empirically (Figure 51).

  • Page 76: The Pinhole And Its Effects

    9.2.5 The pinhole and its effects The reason for deploying confocal microscopy is its ability to create optically thin sections without further mechanical processing of the specimen. The essential component of the instrument that creates these sections is a small diaphragm in front of the detector – the so- called pinhole –...

  • Page 77: Image Detail And Raster Settings

    Figure 53: Optical sections with a variety of pinhole diameters (63x/1.4 objective). pinhole diameters from top left to bottom right: 4 AE; 2 AE; 1 AE; 0.5 AE; 0.25 AE. The strong loss of light can be seen clearly with the small diameters, as can the pronounced background in the images with very large diameters.
  • Page 78 Unlike eyes or conventional cameras, scanners can simply be set to a smaller angle. A further-enlarged section of the field of view will then be displayed on the monitor. It is thus possible to zoom into details without the need for additional optics. As the scan angle can be adjusted very quickly and continuously over a wide range, magnification increases of around 40x can be achieved simply by moving a slider.
  • Page 79 Some purists require 3x oversampling, i.e. 7200 x 7200 pixels, or 52 megapixels. Image formats for x and y can be adjusted independently and in very fine steps, with the Leica TCS SP5 supporting capture formats of up to 64 megapixels (8000 x 8000 pixels) (Table 5).
  • Page 80 Rectangular formats are important for higher image capture rates. An additional parameter is required here: the rotation of the scan field. As field rotation is performed optically in the Leica TCS SP5, rotation by +/- 100° does not have any effect on the speed and possible grid formats (Figure 56).

  • Page 81: Signal And Noise

    Figure 56: Zoom, Pan and Rotation combined in one example. Finally, it must be pointed out in this section that a good microscopic image in a scientific context must always contain a scale. Such a scale can simply be added to the image and adjusted in its form, color and size as required.
  • Page 82 Turn the illumination back on and adjust the gain to prevent distortion. This configuration work is simplified by special color tables such as "Glow-over/Glow-under" – a table that initially uses yellow and red for intensities in steps to indicate the signal strengths.

  • Page 83: Profile Cuts

    The signal-to-noise ratio may be influenced by a number of other factors in addition to illumination intensity: the speed at which data is captured. The actual speed of the scan, which can be adjusted via the horizontal frequency (1 Hz - 1,400 Hz, conventional scanners only), and the averaging process offer additional options for enhancing the signal.

  • Page 84: Multiparameter Fluorescence

    Multiparameter fluorescence In many cases today, specimens are used that contain more than one fluorescent stain. Multiple stains are achieved using hybridization of various linked fragments (fluorescence in situ hybridization, FISH), through differently marked antibodies or with fluorescence proteins with differing spectral properties.
  • Page 85 Figure 60: Simultaneous capture of two fluorescences, in this case excited by a single laser line. The depiction in the colors green and red is arbitrary. Fluorescence and reflection images can also be rendered at the same time. Using another excitation, this merely requires observing a second "emission band"...

  • Page 86: Beam Splitting

    9.3.2 Beam splitting Beam splitting is very easy to describe in AOBS® systems: there is no need to give it any thought. The AOBS automatically switches a narrow band for the selected lines to ensure that the excitation is applied to the specimen. Such bands have a width of around 2nm. Everything else is available to capture the emission.

  • Page 87: Sequential Capture

    The actual objective for effective unmixing is to determine the required coefficients of the matrix. This is also covered by a variety of methods available in the Leica software. It is advisable to experiment a bit to determine the best method for the task at hand. Since all measurement data contains certain error and noise components, there is no perfect recipe for the ultimate truth.

  • Page 88: D Series

    The simplest approach for the user is to determine the coefficients on the basis of the statistical data of the captured images. In this process, the coefficients of the scatter diagrams of both channels are determined using statistical methods. "Hard" and "soft" separation methods are available, leaving the degree of separation at the user's discretion.

  • Page 89: Distances

    9.4.3 Distances The thickness of the optical sections is important when capturing z stacks. If the spacing between the sections is too large (greater than the thickness of the section), this will result in gaps in the data record and a loss of information. A reconstruction then can no longer be calculated correctly.

  • Page 90: Depictions

    9.4.5 Depictions Figure 61: Gallery of a z stack. This "stamp collection" is well-suited for monochrome publications. As mentioned earlier, a three-dimensional image cannot truly be displayed on a two- dimensional monitor. A variety of methods are therefore available for presenting this information.

  • Page 91: Orthogonal Projections

    9.4.5.3 Orthogonal projections A further option for displaying the full range of information (with losses) compressed into two dimensions is to compute projections of the entire series. The most common method is the so-called maximum projection. The brightest value along the z axis is determined for each pixel and entered into the resulting image at this point.

  • Page 92: Rotated Projections

    9.4.5.4 Rotated projections Figure 63: Stereo image of the same 3D data. A bit of practice is required, but this nevertheless represents a worthwhile exercise for any confocal microscope user. The methods described in 9.4.5.3 initially assume that the projection will be performed along the visual axis.

  • Page 93: Scan Speed

    This amounts to measuring the changes in light intensity at a fixed, preselected point in the Leica TCS SP5 with a temporal resolution of 40 MHz (corresponding to 25ns). Naturally, that particular spot in the specimen can be expected to bleach within a very short time.

  • Page 94: Frap Measurements

    Incremental shifts of the emission band can also be used as the basis for an image series. The Leica SP® detector was thus the first instrument with which a spectral image series could be captured using a confocal microscope. Experience has shown that its technology is the most efficient;...

  • Page 95: Combinatorial Analysis

    Combinatorial analysis Many of the methods described above can be combined and deliver new insights in biology, with both fixed and living specimens. The term "multidimensional microscopy" has been coined to describe this form of combinatorial analysis. However, a certain inflation in this regard has become apparent recently.

  • Page 97: Switching Off The System

    Figure 64: Shutting down the computer Next, turn off the switches on the main switch board for the TCS workstation (Figure 65, 5) and the TCS SP5 scanner (Figure 65, 4). The external fan of the argon laser will switch off automatically after several...
  • Page 98 Figure 65: Main switch board The delayed shutdown of the laser cooling system ensures the operational reliability of the TCS SP5 system. 7. Switch off the microscope and any activated fluorescence lamps. If your system features external lasers (IR, UV or others), switch them off in...

  • Page 99: Disposal

    11. Disposal If you have any questions related to disposal please contact the Leica branch office in your country (see Chapter 12 ).

  • Page 101: Contact

    12. Contact If you have any further questions related to your TCS SP5, please contact the Leica branch office in your country. Please refer to the country list below for contact information. If your country is not listed below, please use the area selector at http://www.confocal- microscopy.com.

  • Page 103: Declaration Of Conformity

    13. Declaration of conformity...

  • Page 105: Glossary

    Instrument parameter settings labeled with the letter «L» are predefined by Leica and cannot be changed. User-defined, modifiable instrument parameter settings are stored below «U» in...
  • Page 106 80 % and the reflection values are between 90 % and 95 %. Experiment A file with Leica-specific data format (*.lei) that consists of one ore more individual images or image series. Images recorded with different scan parameters or result images from image processing can be combined here.
  • Page 107 Fluorite objectives Describes a correction class for objectives. Fluorite objectives are semi-apochromatic, i.e. objectives whose degree of correction falls between achromatic and apochromatic. Frame A frame corresponds to the acquisition of a single optical section. For example, if a single optical section is acquired four times (to average the data and to eliminate noise), then frames are created for this optical section.
  • Page 108 Phase visualization The principle of phase visualization as used by Leica is an optimized alternative method to ratiometric displaying. The main area of application is the measurement of ion concentrations in physiology.In contrast with ratiometric procedures, phase visualization obtains more information on the specimen.
  • Page 109 Spherical aberration An optical image distortion conditional on the varying spacing of parallel light rays of the same wavelength from the optical axis.Light rays that travel through outer lens zones have shorter focal distances than rays that travel through the lens center (optic axis). Stokes shift The Stokes shift is a central term in fluorescence microscopy.

  • Page 111: Safety Data Sheets

    15. Safety data sheets The following are safety data sheets from third-party manufacturers.
  • Page 117 Index of figures Figure 1: System components (overview) ................... 11 Figure 2: Dimensions of the TCS SP5 with inverted stand ............12 Figure 3: Dimensions of the TCS SP5 with upright stand ............12 Figure 4: Specimen area of upright and inverted stand .............. 21 Figure 5: Supply unit with main circuit breaker................
  • Page 118 ................69 Figure 45: Use the "Acquire" arrow button to acquire data in all Leica LAS AF applications..70 Figure 46: The "Live" button starts data acquisition in all Leica LAS AF applications....70 Figure 47: Stack acquisition for 3D, time and lambda series ............
  • Page 122 Leica Microsystems CMS GmbH Tel.: +49 (0)621 7028 - 0 Am Friedensplatz 3 Fax: +49 (0)621 7028 - 1028 D-68165 Mannheim (Germany) http://www.leica-microsystems.com Copyright © Leica Microsystems CMS GmbH • All rights reserved Order No.: 156500002 V04...

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