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CLSM technical data

SP5 MP-spectral FLIM CLSM (Room G03.058, Biocenter Martinsried)

Microscope stage: Leica DM6000 CS (upright)

CLSM Ser.No.: 5100000312

Lasers (resent laser power measurements see end of this page):

Diode-Laser 405nm ('violet') 50mW, Coherent (Power Supply 100-240V 405nm Coherent COH405)

Ar-Laser 458, 476, 488, 496, 514nm 100mW, Lasos LGK 7872 ML05 SP5 (Power Supply Ar-Laser Lasos LGN 4000 SP5)

DPSS-Laser 561nm 10mW, Lasos YLK6110T

HeNe-Laser 633nm 10mW, Lasos LGK 7654 (Power Supply 24V HeNe 633nm Lasos LGK 7654)

Ti:Sapphire 'Mai Tai' ~100fs pulse-laser 710-990nm (tunable) with power output 0,4-2 W, depending on wavelength (see graph; power output additionally depends on pump-laser power setting), 80 MHz pulse rate, used for MP (multi-photon) excitation, also in combination with FLIM.

Mai Tai laser power graph


Photomultipliers (PMT):

2x 'standard', 1x HyD and 2x FLIM (please use the 2 standard PMTs for fluorescence intensity imaging, they are better - the FLIM PMTs are less flexible and sensitive, as long as you do not need more than 2 PMTs simultaneously you should ALWAYS use the standard PMTs 3 and 5!). And please remind all the precautions to be taken, when working with the HyD. Always turn down the gain to a minimum before you start operating it.

FLIM setup (Becker&Hickel adaptation):

2x FLIM PMTs; spectral setup (descanned, in scanhead); Positions 2 and 4.

Fluorescence filter cubes in the CLSM: (the GFP and I3 often get exchanged, dependent on usage)

Filter-cubes - excitation, dichroic, emission / used for e.g.
A - UV, BP 340-380, 400, LP 425 / DAPI, Hoechst, AMCA, CW, many more
G/F/P - blue, BP 470/40, 500, BP 525/50 / GFP
I3 - blue, BP 450-490, 510, LP 515 / AcridinOrange, Acridin Yellow, AstrazoneOrange, Auramine, Aurophosphine, BODIPY FL, Calcein, CalciumGreen, C-FDA, Coriphosphine O, DiO, FDA, FITC, Fluo3, NileRed, Phosphine 3R, Rhodamin123
N2.1 - green, BP 515-560, 580, LP 590 / Acid Fuchsin, Acridine Red, Calcium Orange, EthidiumBromide, FastGreen, Feulgen, PropidiumJodide, Rhodamine B, Thiazin red R, TRITC, XRITC, Xylene Orange

Note: we have a set of Leica filter cubes available in the institute, if you have any specific need contact Thomas Ott. CLSM fluorescence filter revolver layout: Position 4 MUST BE LEFT FREE !!!; Position 5 must be "A" (analysator)

Optical performance and technical integrity testing:

FluoCells prepared slide #1 (F36924 shows bovine pulmonary artery endothelial cells (BPAEC) stained with MitoTracker Red CMXRos for labeling mitochondria, Alexa Fluor 488 phalloidin for labeling F-actin and DAPI for labeling the nucleus.

FocalCheck fluorescence microscope test slide #2 (F36913) provides a robust, reproducible method of evaluating the performance of spectral imaging systems, as well as the ability to discriminate closely overlapping spectra. The slide consists of 6 µm–diameter microspheres labeled with a series of spectrally overlapping dyes (Table 23.2).

PS-Speck™ Microscope Point Source Kit contains four different colors of fluorescence microspheres; each microsphere has a diameter of 0.175 +/- 0.005 mm. This exceptionall small diameter coefficient of variation makes the PS-Speck™ microspheres ideal as uniform, subresolution fluorescent point sources for calibrating instrument optics, especially in three-dimensional imaging applications. This kit’s four ready-to-use 1 mL suspensions contain bright, monodisperse microspheres with excitation/emission wavelengths of 360/440 nm (blue), 505/515 nm (green), 540/560 nm (orange) and 633/660 nm (deep red). The PS-Speck™ Kit also includes a mounting protocol for the user’s convenience and sufficient mounting medium to prepare about 100 slides of each.

Software updated to version 2.7.3_build_9723

Laserpower calibrated (29.01.2016):
MP Laser at 800nm without objective at sample-plane: 500mW

UV, VIS and MP overlays calibrated

Resolution calibrated; with 63x oil objektive: 390nm (Z-resolution)

FLIM setup tested and OK

Objectives: 3x UV lenses calibrated, 2x 63x W, 1x63x Oil
We also have a 25x dipping lens with a N.A. of 0,95 with a working distance of 2.2 mm. The lens is IR corrected, and particularly useful in combination with MP excitation (incl. FLIM). Itlis not corrected for UV-excitation (if used with UV, low transparency and potentially chromatic errors might be expected).

DIC tested and OK

Lenses programming checked, OK, as follows: Pos1/D1 Pos2/E
Pos1/K4 Pos2/K6 Pos3/PH1 Pos4/K7 Pos5/PH2 Pos6/EMP Pos7/PH3