Bugnon Instruments

Acquisition systems

Confocal

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation.^[1] Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within an object.

Leica Stellaris 8

Stand	Leica DMi8 CS, Inverted
Illumination	Leica LED 3
Lasers	405nm DMOD (uv) 488nm (blue) 514nm (turquoise) 561nm (green) 638nm (red) 730nm (deep red)
Objectives	HC PL Fluotar 10x/0.30 HC PL APO 20x/0.75 CS2 HC PL APO 40x/1.30 oil CS2 HC PL APO 63x/1.40 oil CS2
Stage	Fully Motorized
Contrast	DIC
CO2 \| T° control	Yes (both)
Incubation type	Stage-top incubator for 35mm dishes or slides (IBIDI)
Detector type	4x HyD S 1x HyD R (for Cy7, Alexa 750nm, …)
Camera	Leica K5 sCMOS 80%QE, 2048×2048, 6.5×6.5um pixels
Optional modules	FRET, FRAP, Lightning, MicroLab
Software	Leica LAS X STELLARIS
Room	B9-150

Zeiss LSM 780 with Airyscan

Stand	Zeiss AxioObserver Z1, Inverted
Widefield Illumination	X-Cite
Lasers	405nm 458nm, 476nm, 488nm, 514nm 561nm 633nm
Objectives	Plan Neofluar 10x / 0.30 NA Plan Apochromat 20x / 0.8 NA Plan Neofluar 40x /0.6 NA Plan Apochromat 40x / 1.30 NA / oil immersion Plan Apochromat 63x /1.40 NA / oil immersion
Stage	Motorized
Contrast	DIC
CO2 \| T° control	No
Detector type	2x GaAsP Quasar detector (spectral imaging) Airyscan (enhanced resolution and SNR)
Optional modules
Software	Zen black 2012
Location	B9-152

Zeiss LSM 900

Stand	Zeiss AxioObserver Z1, Inverted
Widefield Illumination	HXP 120W
Lasers	405nm 488nm 561nm 640nm
Objectives	Plan Apochromat 10x \| 0.45 NA Plan Apochromat 20x \| 0.80 NA Plan Apochromat 40x \| 1.30 NA \| oil immersion Plan Apochromat 63x \| 1.40 NA \| oil immersion
Stage	Motorized
Contrast	DIC
Cubes	FL set 10 (ExBP450-490 shift free) FL set 20 (Rhodamin shift free) FL set 49 (DAPI, ExG365 shift free)
CO2 \| T° control	No
Detector type	3x GaAsP 1x ESID (bright field)
Optional modules	Tiles and Positions FRAP Experiment Designer
Software	Zen blue 2019
Location	B9-152

Zeiss LSM 900 BSL-2 Lab [SPECIAL AUTHORIZATION REQUIRED]

This particular setup being placed in a BSL-2 (Bio Safety Level 2) Lab, a special authorization is required to access it.
The management and access to this BSL-2 confocal microscope is under the direct supervision of IMUL (contact: Jérôme Gouttenoire, Jerome.Gouttenoire@chuv.ch, + 41 79 556 60 93 – or- Nicolas Jacquier, Nicolas.Jacquier@chuv.ch, +41 21 314 8539

Stage	Zeiss AxioObserver 7, Inverted
Illumination	HXP 120V
Lasers	405nm 488nm 561nm 640nm
Objectives	EC Plan Neofluar 2.5x / 0.085 NA Plan Apochromat 10x / 0.45 NA DIC II Plan Apochromat 20x / 0.80 DIC II Plan Apochromat 40x / 0.95 DIC III Plan Apochromat 40x / 1.30 NA / oil immersion DIC II Plan Apochromat 63x /1.40 NA / oil immersion DIC II Plan Apochromat 100x /1.40 NA / oil immersion DIC II
Stage	Motorized XY Z-Piezo Definite Focus 2
Contrast	DIC for 20x, 40x, 63x and 100x
CO2 \| T° control	Yes Full enclosure
Detector type	2x GaAsP Airyscan 2 T-PMT for transmitted light
Optional modules	Multiposition and Tiling
Software	Zen Blue 2.6 Measurement, Multi channel, Panorama, Extended Focus, Image analysis, Time lapse, Z stack, Autofocus, Colocalization, Spectral unmixing, Connect entry, 3Dxl, Tiles&positions, sample navigator and FRAP analysis
Room	BU48/04/402 (CHUV)

Spinning Disk | Time-Lapse

Spinning-disk (Nipkow disk) confocal microscopes use a series of moving pinholes on a disc to scan spots of light. Since a series of pinholes scans an area in parallel, each pinhole is allowed to hover over a specific area for a longer amount of time thereby reducing the excitation energy needed to illuminate a sample when compared to laser scanning microscopes. Decreased excitation energy reduces phototoxicity and photobleaching of a sample often making it the preferred system for imaging live cells or organisms.

Nikon Ti2 | CrEST Optics X-Light V3

Stand	Nikon Ti2, Inverted
Spinning Disk Head	Crest Optics X-Light V3 – Single Pattern Disk for large FOV
Widefield LED Illumination	Lumencor Sola LED
Filter Cubes	DAPI CFP YFP GFP CY3 CY5
Spinning Disk + DMD LED excitation	Spinning Disk: Lumencor Celesta 408nm 445nm 473nm 518nm 545nm 635nm 750 nm DMD: Lumecor Spectra X 395nm 440nm 470nm 508nm 555nm 640nm
Objectives	CFI Plan Apochromat Lambda 10X, N.A. 0.45, W.D. 4.0mm CFI Plan Apochromat Lambda 20X, N.A. 0.75, W.D. 1.0mm, spring-loaded CFI Plan Apochromat Lambda 40XC N.A. 0.95, W.D. 0.21mm, Spring-loaded, Cover glass correction: 0.11-0.23mm CFI Plan Apochromat Lambda 60XC N.A 0.95, WD 0.21mm CFI Plan Apochromat Lambda 60X Oil, N.A1.40, W.D. 0.13mm
Stage	Motorized Piezo (Z direction) Nikon PFS (Perfect Focus System)
Contrast	DIC
CO2 \| T° control	Yes [Full enclosure]
Cameras	1x Photometrics Prime 95B 1x Nikon DS-Qi2
DMD	DMD module for multipoint photoactivation
Optional modules	JOBS General Analysis 3
Software	Nikon NIS-Elements AR 5.0
Room	B9-145c

Widefield | Fluorescence

The majority of fluorescence microscopes, especially those used in the life sciences, are of the epifluorescence design shown in the diagram. Light of the excitation wavelength illuminates the specimen through the objective lens. The fluorescence emitted by the specimen is focused to the detector by the same objective that is used for the excitation which for greater resolution will need objective lens with higher numerical aperture. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light and the epifluorescence method therefore gives a high signal-to-noise ratio. The dichroic beamsplitter acts as a wavelength specific filter, transmitting fluoresced light through to the eyepiece or detector, but reflecting any remaining excitation light back towards the source.

Leica DMi8

Stand	Leica DMi 8, Inverted
Illumination	SOLASMII, Light source (365nm Version)
Filters	DAPI YFP CFP CY3 Y5 GFP
Objectives	HCX FL PLAN 5/0,12 working distance 14,2mm HC PL Fluotar 10x/0.32 PH1 HC PL FL L 20x/0.40 CORR PH1 HCX PL FLUOTAR L 40x/0.60 CORR HC PL Fluotar (340) 40x/1.30 oil HC PL APO 63x/1.400.60 oil
Stage	Motorized
Contrast	Phase
CO2 \| T° control	No
Cameras	Color camera Leica DFC7000T Black and White Camera Andor Zyla
Optional modules	Navigator Multi-well holder (Universal holding frame KM, Click-In)
Software	LAS X Premium
Room	B9-152c

Stereomicroscopy

The stereo, stereoscopic or dissecting microscope is an optical microscope variant designed for low magnification observation of a sample, typically using light reflected from the surface of an object rather than transmitted through it. The instrument uses two separate optical paths with two objectives and eyepieces to provide slightly different viewing angles to the left and right eyes. This arrangement produces a three-dimensional visualization of the sample being examined.^[1] Stereomicroscopy overlaps macrophotography for recording and examining solid samples with complex surface topography, where a three-dimensional view is needed for analyzing the detail.

Nikon SMZ25 Stereomicroscope | Macroscope

Stand	Nikon SMZ-25, Upright
Illumination	Bright field from the base Fluorescence Ring illumination module for macro shots Optional screw-in polarizer
Filters	DAPI GFP TXR
Objectives	Plan Apochromat 0.5x SHR WD 71mm Plan Apochromat 1x SHR WD 60mm Plan Apochromat 2x SHR WD 20mm Motorized 25x zoom
Stage	Manual XY Motorized Z
Contrast	Brightfield + Fluorescence
CO2 \| T° control	No
Cameras	Nikon DS-Ri2 16 Mpx color camera
Optional modules	Extended Depth of Field JOBS Multi-position
Software	Nikon NIS-BR 4.20
Room	B9-152

Slide scanner

A slide scanner allows you to digitize full microscopy slides in an automated manner. This is a high througput system able to process around a hundred of slides in one batch.

Zeiss Axioscan Z.1

The way the Zeiss slide scanner works at the CIF implies that designing a specific profile for each of your sample type is needed.

This profile is very dependent of the way you have prepared your slides. Since this processe is time-consuming, it is only useful if you have at least a dozen or more slides of the exact same type to acquire (1-2 hours with testing).

Here are some general guidelines before thinking of using the slide scanner for your experiments:

*SCAN PROFILE*
*If the samples vary in any one of these categories:* Thickness of the slices Layout of the cuts Size of the cuts Intensity / color of the staining *Fluorescence specific:* Number of fluorescent dyes target of the staining used as reference *The profile will have to be updated* and saved as a new variant or it will probably fail (not focused, over/underexposed, stitching failing).
*ACQUISITION TIME*
*The acquisition time depends on:* Number of sample / blade Sample size / blade Lens used Exposure time (not important in BF) Z Stack or not and EDF(extend focus) or not Accuracy of the two focus maps Each of these parameters can greatly vary the acquisition time, which can range from a few minutes to an hour or more per slide. If you want to change the objective used for the acquisition, you will also need to update most of the previous parameters of the scan profile.
*DATA SETS*
*The files are acquired in the .CZI format.* The typical file size ranges from 100 Mb to several Gb Files exported in TIFF will see their size increased AND multiplied several times depending on the number of channels It is thus key to minimize the size of the dataset to be acquired to avoid further analysis problems -> always prefer the minimum magnification that will answer your scientific question. For screening purposes, use the lowest magnification.

Illumination	Zeiss Colibri 7 Type R[G/Y]CBV-UV Red (630nm) for excitation of Cy5, Alexa 631, TOTO-3 and similar dyes Yellow (590nm) for excitation of mCherry, Alexa 568, mPlum and similar dyes Green (555nm) for excitation of Cy3, TRITC, DsRed and similar dyes Cyan (511nm) for excitation of eYFP, Eosin, TOTO-1 and similar dyes Blue (475nm) for excitation of eGFP, Fluo4, FITC and similar dyes Violet (430nm) for excitation of eCFP, Lucifer Yellow, Alexa 430 and similar dyes UV (385nm) for excitation of DAPI, Alexa 405, Hoechst 33258 and similar dyes
Filters	BFP : EX BP 390/40, BS FT 420, EM BP 450/40 96 HE BFP shift free (E) DAPI : 387/11, 409, 447/602 F36-513 eGFP : 470/40, 495, 520 / 50 38 HE eGFP shift free (E) Cy3 : 550/25, 570, 605/70 43 HE Cy 3 shift free (E) mPlum : 587/25, 605, 647/70 64 HE mPlum shift free (E) Cy5 : 640/30, 660, 690/50 50 Cy 5 shift free (E)
Objectives	Fluar 5x/0.25 Plan Apochromat 10x/0.45 Plan Apochromat 20x/0.80 Plan Apochromat 40x/0.95
Stage	Motorized
Contrast	Brightfield, RAC, Fluorescence
CO2 \| T° control	No
Cameras	Color camera 3CCD Hitachi HV-F202SCL for Axio Scan.Z1 Black and White Camera Orca Flash 4.0 V2 for Axio Scan.Z1
Optional modules
Software	Zen Blue 2.6
Room	B9-152c

Image Processing

[Under construction]