Operating the Confocal Microscope

Operating The LeicaTCS-NT Confocal Microscope

0. Notices

1. Principles of Confocal Scanning

2. General Running of the Confocal System

2.0 Know the specimen under conventional microscopy! ^*
2.1 Check list guide of basic operation
2.1.1 Power up the confocal system
2.2 Setup the microscope for conventional viewing - Kohler illumination
Find the specimen
2.1.3 Setup the microscope for confocal scanning
2.1.4 Setup the computer for scanning
2.1.4 Saving scanned images
2.1.5 Shut down and clean up the confocal system. Powering down the system in an orderly and correct sequence is important especially for the lasers and mercury lamp, which are stressed during the powering down and up cycles. If you have booked within 3 hours or less after the previously booked user you are responsible for ensuring that the system is powered downed especially if you have cancelled. Contact information is provided on the booking calendar page. If you doubt that next user will appear, please power down the system, especially near evening time. Your effort and diligence is highly appreciated!
2.3 FAQs - frequently asked questions with answers or getchuas

3. Special Scanning Operations ^*
(Note this section will not be updated until the new Leica scanning software is installed after 2000)

3.1 Multi-channel simultaneous scanning ^*
3.2 Multi-channel sequential scanning
3.3 Transmitted light images while laser scanning ^*
3.4 Differential Interference Contrast and confocal images ^*
3.5 Reflectance mode ^*
3.6 Reflectance and fluorescence time lapse scanning ^*
3.7 XZ Scanning - a side on view ^*
3.8 Z-section series ^*
3.9 Extended focus - first collect a z-section series then generate extended focus image ^*
3.10 Time-series ^*
3.11 Fluorescence recovery after photo bleaching - FRAP ^*
3.12 Fluorescence resonance energy transfer - FRET ^*

4. Advanced Scanning Considerations ^*

4.1 Pinhole Size - Confocal Aperture
- Depth of Focus
- Resolution
- Light Transmission
- Noise
4.2 Laser Power ^*
- Noise
- Photo bleaching
- Phototoxicity
4.3 Sensitivity ^*
- Laser Power - 1. AOTF - 2. Main knob on Krypton and Argon lasers
- Pinhole size
- Objective NA and magnification (and that objective iris)
- Gain and offset of Photo-Multiplier-Tube (PMT)
- Scanning speed
- Dye concentration and conditions
- Fluorescence saturation
- Quenching
- Photo bleaching
- Path absorbance
- Scanning dwell time
4.4 Multi-channel Scanning ^*
- bleed through - cross talk - emission coupling
- sequential scanning

5. Image processing with LeicaTCS-NT

Acquire:	5.1 Making an overlayed Red/Green/Blue (RGB) image from a "file -> save as" image
View:	5.2 Viewing z-series stacks or time-series images sequentially
View:	5.3 Making an extended focus image and saving it using "file -> save selected")
Pseudo 3-D:	5.4 Orthogonal viewing of z-series stacks or time-series image
3-D:	5.5 3D rendering - maximum brightness projection

6. Handling Leica Image Files on PCs

6.1 Tiff image file format - single channel - multi channel - RGB - stacks
6.2 Splitting z-series or time-series or multi-channel tiff files into individual tiff files using tiffsplit4.exe
6.3 Making an extended focus image from a z-series file - using tiffsplit4.exe
6.3.2 Making maximum intensity projections using tiffsplit4.exe (version 4.10 or later)
6.4.1 Overlaying images into red & green using PhotoShop - tedious, but it works ^*
6.4.2 Overlaying a fluorescence image on a gray scale image using PhotoShop. ^*
6.5 Adding scale bars, post hoc, with PhotoShop ^*
6.6 2D Spatial filtering, removing noise, enhancing edges ^*

7. Image Processing Bitmap Image Files on PCs

7.1 Making bitmap image files from tiff files
7.2 3-D Rendering & perspective rotation using Slicer / T3D
7.3 Making 3-D stereo pairs with Slicer / T3D
7.4 Overlaying a series of bitmap images into red & green bitmap files using bmovrlay.exe ^*
7.5 Making an extended focus image from a z-series using bmpsum.exe - or using tiffsplit4.exe
7.6 Animation / movie production from time lapse, z-series or 3-D rotation image data ^*
7.7 Co-localization - red/green overlap issues: beware! ^*
7.8 Co-localization - image multiplication using bmproduct.exe ^*
7.9 Resizing and rescaling images for transfer to Medical Illustrations ^*
7.10 bmfunctions : general command line programs for manipulation of bitmap file series, z-series or time-series - must convert images from tiff files (.tif) into bitmap files (.bmp) to use these command line programs ^*

8. Image Processing Using Confocal Assistant on PCs

8.1 Using Confocal Assistant an overview
8.2 Converting Leica Tiff image format into Biorad PIC Image format for use with Confocal Assistant
8.3 Processing Image z-series/time-series stacks in Confocal Assistant
- 8.3.1 Starting Confocal Assistant and viewing images - single images - PIC z-stacks/t-series
- 8.3.2 Animating a PIC image series - Save a modified PIC image series
- 8.3.3 Making projections
- 8.3.4 Making 3D views, static stereo pairs and red/green anaglyphs
- 8.3.5 Making dynamic 3D views - animated, rocking views
- 8.3.10 Other operations that may be performed by Confocal Assistant
8.4 Converting a PIC format image file into a bitmap series (if you have to)

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Last updated 07-31-2003

					© 1998-2003 Michael Chua, Cell & Molecular Physiology, UNC. All rights reserved.
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