Scanning with Mercury Lamp
	Examples for scanning with mercury lamp for UV excitation and more... enlarge Endothelial cells, triple FL (nuclei - DAPI, mitochandria - Mitotracker Red, F-actin - BODIPY) Objective PlanApochromat 63x/1.4 Oil Scan speed 8 (325 Hz line frequency) Average mode 8x line Pinhole sizes 1 Airy (1.5 Airy for DAPI channel) Image resolution 512 x 512 x 3 ch x 12 Bit Illumination HBO 103, VIS lasers (488, 543 nm) Detection Multitracking mode enlarge Mouse intestine section, triple FL (nuclei stained with Alexa 350) Objective PlanNeoFluar 20x/0.5 Scan speed 9 (520 Hz line frequency) Average mode 16x line Pinhole size 1 Airy (1.5 Airy for UV channel) Image resolution 512 x 512 x 3 ch x 8 Bit Illumination HBO 103, VIS lasers (488, 543 nm) Detection Multitracking mode		back
	General comment: UV excitation using HBO is a feasible way to localize DAPI stained nuclei within cells and tissue. However, using UV laser (available for LSM 510) is certainly superior in terms of confocality, operation, attenuation and reduced amount of bleaching and photo toxicity. System setup: Triple fluorescence images recorded with standard LSM 5 PASCAL on Axiovert 200 M using standard DC controlled HBO103 mercury lamp (stability +/-1%) for blue channel and the laser lines 488 nm and 543 nm for green and red channels. HBO 103 connected to standard FL illumination port. Acquisition of DAPI channels is half confocal (bright field illumination and point scan detection). 3D scans of DAPI images are feasible. The Multitracking feature of the LSM was used to prevent emission crosstalk. For image #2: all 3 single channels scanned by turns and automatically copied into multicolor image. For image #1: red/green channels scanned in Multitracking mode first to avoid HBO bleaching, followed by separate DAPI channel scan. Channels digitally merged using Copy function. Setup of DAPI channel: DAPI filter cube inserted into the fluorescence filter revolver of microscope at (normally free) LSM position. PMT Ch1 used for DAPI channel (emission filter, primary and secondary beam splitter of LSM set to ‘none’ position). DAPI filter cube serves to reflect UV excitation and transmit DAPI emission (long pass characteristics of filter allow VIS laser excitation and detection standard LSM way, but with lower transmission).
	More ways to use the LSM: Reflectance Confocal Microscopy: enlarge Ultra-thin section of rat colon embedded in Spurr-resin stained with Toluidin Blue Objective PlanApochromat 63x/1.4 Oil Scan speed 7 (260 Hz line frequency) Pixel time 1.12 µs Average mode 4x line Pinhole sizes < 1 Airy Image resolution 1024 x 1024 x 3ch x 8 Bit Illumination Red, green, blue lasers Detection Internal PMTs, Multitracking Using the LSM as an RGB camera (1) enlarge Young mouse section histological staining Objective PlanNeoFluar 10x/0.3 Scan speed 8 (325 Hz line frequency) Pixel time 0.4 µs Average mode none Pinhole size <1 Airy Image resolution 2048x2048 x 3ch x 12 Bit Illumination Red, green, blue lasers Detection Transmission PMT, Multitracking Using the LSM as an RGB camera (2) enlarge Young mouse section histological staining Objective PlanNeoFluar 10x/0.3 Scan speed 8 (325 Hz line frequency) Pixel time 0.88 µs Average mode 16x line Pinhole size 1 Airy Image resolution 1024 x 1024 x 3ch x 8 Bit Illumination halogen lamp (tricky) Detection Internal PMT, Multitracking
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