Zobrazovací metody

Zobrazovací metody

Mgr. Aleš Benda, Ph.D.

Mgr. Aleš Benda, Ph.D. — Vedoucí laboratoře

O nás

Servisní laboratoř Zobrazovací metody pokrývá tři základní oblasti – optickou (fluorescenční) mikroskopii, elektronovou mikroskopii a průtokovou cytometrii. Nabízíme našim uživatelům nejen zacvičení a přístup k pokročilým přístrojům, ale i konzultační služby týkající se optimálního nastavení podmínek experimentu, přípravy vzorku, sběru dat, případně analýzy a interpretace získaných výsledků.

Napojení na národní a mezinárodní výzkumné infrastruktury a konsorcia

Euro-Bioimaging

Servisní laboratoř Zobrazovací metody tvoří část pražského uzlu celoevropské zobrazovací infrastruktury Euro-BioImaging.

http://www.eurobioimaging.eu/

 

Czech-Bioimaging

Provoz servisní laboratoře je částečně hrazen z prostředků programu pro velké výzkumné infrastruktury Ministerstva školství, mládeže a tělovýchovy v rámci projektu „Národní infrastruktura pro biologické a medicínské zobrazování (Czech BioImaging – LM2015062)“.

https://www.czech-bioimaging.cz/

 

 

Rezervační systém

 

Umístnění v centru BIOCEV a plán místností IMCF

Plán místností v servisní laboratoři 

 

Aktuální stav a historie teplot v jednotlivých místnostech servisní laboratoře

 

Aktuality

Akce

Služby

Naše servisní laboratoř poskytuje služby v oblasti optické (fluorescenční) mikroskopie, elektronové mikroskopie a průtokové cytometrie

How to access our services

Information for new users:

  1. Contact us
    • If you want to use our services, do not hesitate to call or email anyone from our team

  2. Meet us for the first time
    • A meeting will be scheduled for you to meet with IMCF staff members. Here we get to discuss your project and see how we can best assist you.

  3. Register to booking system
    • In order to keep track of the instrument usage we use booking system. It also allows you to readily manage your reservations. Steps for registration are outlined here.

  4. Attend theoretical and practical training
    • Few of our instruments are simply push button systems, and as such we do our best to help you understand fundamentally how they function so you can make informed decisions. 

  5. Recieve a certificate
    • After attending the training on given instrument you get the certification that allows you to use the instrument unsupervised.

  6. Independent measurement
    • Now you can use the instrument on your own. Beware that IMCF staff is here for you, so do not hesitate to ask for help.

Služby

01

Optická (fluorescenční) mikroskopie

02

Elektronová mikroskopie

03

Průtoková cytomerie

Vybavení

Elektronová mikroskopie

Leica EM ACE600 – coating system

Leica EM ACE600 – coating system

High Vacuum Sputter Coater

Application

  • Metal coating of sample to cerate a conductive layer which inhibits charging, reduces thermal demage and improves the secondary electron signal in the SEM
  • Making the carbon layers to be transparent to the electron beam but conductive (needed for X-ray microanalysis), to support films on grids
  • Glow discharge to render grids surface hydrophilic
  • Freeze-Fracture of frozen samples

Main components

The Leica EM ACE600 includes the following main functional units:

  • High vacuum chamber
  • Rotation sample stage, 24 positions for 12,7 mm SEM stubs
  • Quartz thickness measurement
  • Carbon thread source or sputter source (platinum and gold targets)
  • Freeze-Fracture cryo set includes vacuum cyro transfer Leica EM VCT100 and cryo stage
Leica EM CPD300 – critical point dryer

Leica EM CPD300 – critical point dryer

Automated Critical Point Dryer 

Application

  • The drying of biological specimens such as pollen, tissue, plants, insects for SEM analysis
  • The drying of industrial samples , for example MEMS (Micro Electro Mechanical Systems

Method

Crytical point drying is a method which use of CO2 as transitional fluid because its critical point is at 31 °C and 73,8bar. Technically these temperature and pressure requirements of the CO2 can be implemented relatively easily then for water (critical point is 374 °C and 228,5bar). The water in the cell is replaced by an acetone which is very soluble with liquid CO2. This procedure is follow by substitution with liquid CO2 through serial dilution steps. The increasing the temperature and pressure will transfer the CO2 through its critical point into a subcritical state. By controlled depressurization and constant temperature is subcritical CO2 convert into its gaseous phase without crossing the phase boundary between liquid and gas. This technique of preparing sample for scanning electron microscope is more gentle and better preserve structure than air drying sample.

Leica EM FSP - freeze substitution processor

Leica EM FSP - freeze substitution processor

an automatic reagent handling system for AFS2

Leica EM AFS2 – automatic freeze substitution

Leica EM AFS2 – automatic freeze substitution

cryo-substitution for samples frozen in HPM100

Leica EM HPM100 – high pressure freezing machine

Leica EM HPM100 – high pressure freezing machine

cryofixation of samples for EM

FEI Helios NanoLab 660 G3 UC

FEI Helios NanoLab 660 G3 UC

Scanning electron microscope with focused ion beam milling equipped for cryo-imaging and correlative light and electron microscopy (CLEM)

Application

  • 3D scanning electron microscopy (FIB-SEM)
  • high resolution scanning electron microscopy (embedded, dried or frozen samples)
  • elemental analysis (EDS)

More info

Průtoková cytometrie

BD LSRFortessaTM SORP

BD LSRFortessaTM SORP

The BD LSRFortessaTM SORP is a benchtop digital flow cytometer. Equipped with 5 spatially separated lasers, 18 fluorescent detectors and 2 light scatter detectors for fast multicolour analysis with accurate data acquisition rate at up to 40,000 events per second (with beads). The BD LSRFortessaTM has a BDTM High Throughput Sampler (HTS) for increased lab productivity by acquiring samples from a 96- or 384-well plates.

Applications

  • Multicolour flow cytometry analysis.

More info

BD FACS Aria Fusion

BD FACS Aria Fusion

Fluorescence activated cell sorter (FACS) with a quartz cuvette flow cell that is in fixed alignment with the laser, and is gel coupled to the collection optics. BD FACSAriaTM Fusion is equipped with 5 spatially separated lasers, 18 fluorescent detectors and 2 light scatter detectors for fast multicolour analysis and cell sorting with accurate event data acquisition at up to 70,000 events per second. BD FACSAriaTM Fusion is placed in the biosafety cabinet (BSC) Class II Type A2, designed in collaboration with The Baker Company, a leader in biosafety solutions.

Applications

  • Flow cytometry analysis and fluorescence activated cell sorting.

 Excitation Optics

Fixed optical alignment of all Class IIIb lasers with the cuvette flow cell. All lasers are solid state and elliptical:

Beam height: 9 ±3 µm

Beam width: 65 ±7 µm

 

More info

Optická mikroskopie

Abberior Instruments STED

Abberior Instruments STED

Fluorescence inverted confocal microscope and STED super-resolution nanoscope with high detection efficiency of far-red photons. The system is equipped with four excitation lasers, depletion laser 775 nm and four single photon counting detectors. For more detailed microscope characteristics please see the section “Microscope”.

Basic introduction to STED microscopy

Application

  • Two-color 2D and 3D super-resolution images obtained by STED technique with a pulsed depletion laser 775 nm and pulsed excitation 561 nm and 640 nm
  • STED RESCue mode available (STED imaging mode that significantly reduces the light dose sent onto the sample without compromising the resolution)
  • 2D and 3D STED images available also with water immersion objective
  • Multicolor confocal scanning system with variable pinhole size
  • Possible FLIM or FCS acquisition
  • Live cell imaging available

More info about the instrument

Leica DMi8 WF

Leica DMi8 WF

Inverted widefield fluorescence microscope equipped with epifluorescence lamp and EM-CCD camera.

Application

  • Fast and multi-color widefield imaging
  • Various measurement options: z-stack, time series, tile scan, multi positions
  • Brightfield and DIC microscopy

More info

Leica TCS SP8 MD6 CFS

Leica TCS SP8 MD6 CFS

An upright fluorescence confocal microscope equipped with four excitation lasers, dipping objectives with long working distance, oil immersion objective and 1x HyD detector and 2x PMTs. Presence of camera, epifluorescence lamp and removable infrared filter allows also widefield imaging in infrared area. Accessory for electrophysiological experiments is also available.

Application

  • Fast and multi-color confocal imaging
  • Fast and multi-color widefield imaging
  • Various measurement options: z-stack, time series
  • Spectral imaging, measurement of emission spectrum
  • Option of infrared detection in widefield imaging
  • Brightfield and polarized light microscopy

More info

Super-resolution Nikon Ti-E microscopes with N-SIM and N-STORM

Super-resolution Nikon Ti-E microscopes with N-SIM and N-STORM

Multifunctional fluorescence inverted widefield microscope enabling live-cell imaging, TIRF or HILO illumination and two super-resolution techniques: structured illumination microscopy and single molecule localization microscopy. For more detailed microscope characteristics please see the section “Microscope”.

Basic introduction to super-resolution microscopy

Application

  • Multicolor (up to 3 colors) super-resolution images obtained by 3D-SIM technique reaching up to 2x better lateral and axial resolution compared to standard widefield imaging
  • Super-resolution 2D images at high speed captured by „TIRF-SIM“ mode for excitation wavelengths 488 and 561 nm for better understanding of molecular interactions at the cell surface
  • Two-color super-resolution images obtained by single molecule localization methods (STORM, dSTORM, PALM; TIRF or HILO illumination available) with resolution improvement up to 10x compared to conventional optical microscopes
  • Fast and sensitive multicolor widefield imaging with TIRF or HILO excitation option
  • Option of simultaneous dual wavelength imaging by single camera
  • Long term live-cell imaging available
  • Brightfield microscopy

More info

Nikon H-TIRF

Nikon H-TIRF

Versatile fluorescence inverted widefield microscope equipped with four lasers, epifluorescence lamp and EM-CCD and CMOS cameras. The microscope is designed for short and long live-cell imaging and experiments under standard or TIRF illumination.

Link to manufacturer website

 Application

  • Fast and sensitive multicolor widefield imaging with TIRF, HILO or EPI excitation option
  • Option of simultaneous dual wavelength imaging by single camera
  • Short or long term live-cell imaging available
  • Various measurement options: z-stack, time series, tile scan, multi positions
  • Brightfield and DIC microscopy

More info

Leica TCS SP8 WLL SMD-FLIM

Leica TCS SP8 WLL SMD-FLIM

Versatile inverted fluorescence confocal microscope equipped with 405 nm, 445 nm and white light laser, spectrally tunable detection and FLIM hardware. Presence of camera and epifluorescence lamp allows also widefield imaging. For more detailed microscope characteristics please see the section “Microscope”.

Application

  • Fast and multi-color confocal imaging
  • Fast and multi-color widefield imaging
  • Various measurement options: z-stack, time series, tile scan, multi positions
  • Spectral imaging
  • Measurement of emission and excitation spectra
  • Lifetime imaging, FLIM-FRET
  • Measurement of e.g. membrane dynamics by FCS and FRAP
  • Live-cell imaging available
  • Brightfield, DIC, polarized light microscopy

More info

Carl Zeiss LSM 880 NLO

Carl Zeiss LSM 880 NLO

Intravital inverted two-photon and confocal microscope with 34 channel spectral detection and with four reflected and two transmitted NDD detectors (including two highly sensitive GaAsP detectors suitable for FLIM), equipped with MP laser with OPO and with the full set of visible cw lasers.

More info:

Carl Zeiss AxioObserver.Z1 with confocal module LSM 880 NLO and MP excitation

Description

LSM 880 NLO offers many imaging modes – except the classic scanning confocal mode, the system is equipped by 32-channels detector for spectral detection and software to separate probes with very similar spectra (e.g. GFP and YFP) and also by multiphoton laser with OPO, so tuning of wavelengths in range 690 nm – 1300 nm is possible. The part of the multiphoton light path is also motorized periscope for fine adjusting/focusing of MP beam into the scanning head of the microscope. Detection part of multiphoton light path contains NDD.2 module with 2 standard PMT detectors and BIG.2 module with 2 GaAsP PMT detectors. The system has wide range of 1P excitation lasers – 405, 458, 488, 514, 561 and 633 nm, epifluorescence light source (metal halide lamp) and GFP and RFP fluorescence filter sets for eyepiece detection of signal, wide range of objectives – 5x, 10x, 25x, 40x, 63x water immersion, 63x oil immersion. For detection of transmitted light, the microscope has another NDD.2 module with 2 detectors. The source for transmitted light is VIS-LED. Insert with controlled temperature is also available with the system.

Whole system is controlled by ZEN Black with 3D visualization, FRAP Efficiency, Tiles and Positions, Experiment designer license keys.

 

Applications:

  • Deep imaging of thick biological samples due to the multiphoton excitation
  • Spectral detection, spectral unmixing and fingerprinting
  • Confocal imaging
  • FLIM
  • SHG, THG

 

Lasers:

405nm, CW, 30mW

Argon laser: 458, 488, 514nm, 25mW

561nm, 20mW

633nm, 5mW

 

Objectives:

EC Plan-Neofluar 5x, NA 0.16, WD 18.5mm

Plan-Apochromat 10x, NA 0.45, WD 2.1mm

LD LCI Plan-Apochromat 25x Correction collar, NA 0.8, multi-immersion: water, oil, glycerin, WD0.57 at D=0.17 (correction for coverglass thickness D=0-0.17), DIC

LD C-Apochromat 40x Correction collar, NA 1.1, water immersion, D=0.14-0.19, WD0.62 at D=0.17, DIC

C-Apochromat 63x Correction collar, NA 1.2, water immersion, D=0.14-0.19, WD0.28 at D=0.17, DIC

Plan-Apochromat 63x, NA 1.4, oil immersion, WD 0.19mm, DIC

 

Epifluorescence filter sets

In system:

  • Filter set GFP: Ex BP 470/40, BS FT 495, EM BP 525/50
  • Filter set Cy3: Ex BP 545/25, BS FT 570, EM BP 605/70

 

Out of the system:

  • Filter set DAPI: EX G 365, BS FT 395, EM BP 445/50
  • Filter set Cy5: Ex BP 640/30, BS FT 660, EM BP 690/50

Publikace

2018

Font-Haro, A. et al. Expression of TIM-3 on Plasmacytoid Dendritic Cells as a Predictive Biomarker of Decline in HIV-1 RNA Level during ART. Viruses 3, 1–13 (2018).

Agarwal, K., Macháň, R. & Prasad, D. K. Non-heuristic automatic techniques for overcoming low signal-to-noise-ratio bias of localization microscopy and multiple signal classification algorithm. Sci. Rep. 8, 4988 (2018).

2017

Zackova Suchanova, J., Neburkova, J., Spanielova, H., Forstova, J. & Cigler, P. Retargeting Polyomavirus-Like Particles to Cancer Cells by Chemical Modification of Capsid Surface. Bioconjug. Chem. 28, 307–313 (2017).

Horníková, L., Fraiberk, M., Man, P., Janovec, V. & Forstová, J. VP1, the major capsid protein of the mouse polyomavirus, binds microtubules, promotes their acetylation and blocks the host cell cycle. FEBS J. 284, 301–323 (2017).

Braniš, J. et al. The role of focal adhesion anchoring domains of CAS in mechanotransduction. Sci. Rep. 7, (2017).

Voleman, L. et al. Giardia intestinalis mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum. BMC Biol. 15, (2017).

Skultetyova, L. et al. Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules. Sci. Rep. 7, (2017).

Kellerová, P. & Tachezy, J. Zoonotic Trichomonas tenax and a new trichomonad species, Trichomonas brixi n. sp., from the oral cavities of dogs and cats. Int. J. Parasitol. 47, 247–255 (2017).

Kokavec, J. et al. The ISWI ATPase Smarca5 (Snf2h) Is Required for Proliferation and Differentiation of Hematopoietic Stem and Progenitor Cells. Stem Cells 35, 1614–1623 (2017).

Danko, M., Hrdlovič, P., Martinická, A., Benda, A. & Cigáň, M. Spectral properties of ionic benzotristhiazole based donor-acceptor NLO-phores in polymer matrices and their one- and two-photon cellular imaging ability. Photochem. Photobiol. Sci. 16, (2017).

Huérfano, S., Ryabchenko, B., Španielová, H. & Forstová, J. Hydrophobic domains of mouse polyomavirus minor capsid proteins promote membrane association and virus exit from the ER. FEBS Journal 284, 883–902 (2017).

Kostrouchová, M. et al. The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development. PeerJ 2017, (2017).

Luo, W. et al. ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility. J. Cell Sci. 130, 2382–2393 (2017).

Blecha, J. et al. Antioxidant defense in quiescent cells determines selectivity of electron transport chain inhibition-induced cell death. Free Radic. Biol. Med. 112, 253–266 (2017).

Danko, M., Hrdlovič, P., Martinická, A., Benda, A. & Cigáň, M. Spectral properties of ionic benzotristhiazole based donor-acceptor NLO-phores in polymer matrices and their one- and two-photon cellular imaging ability. Photochem. Photobiol. Sci. 16, (2017).

Kostrouchová, M. et al. The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development. PeerJ 2017, (2017).

2016

Boukalova, S. et al. Mitochondrial Targeting of Metformin Enhances Its Activity against Pancreatic Cancer. Mol. Cancer Ther. 15, 2875–2886 (2016).

Dvorakova, M. et al. Incomplete and delayed Sox2 deletion defines residual ear neurosensory development and maintenance. Sci. Rep. 6, 1–16 (2016)

Ma, Y., Benda, A., Nicovich, P. R. & Gaus, K. Measuring membrane association and protein diffusion within membranes with supercritical angle fluorescence microscopy. Biomed. Opt. Express 7, (2016).

Benda, A., Aitken, H., Davies, D. S., Whan, R. & Goldsbury, C. STED imaging of tau filaments in Alzheimer’s disease cortical grey matter. J. Struct. Biol. 195, (2016).

Jiang, Y. et al. Time-resolved fluorescence anisotropy study of organic lead halide perovskite. Sol. Energy Mater. Sol. Cells 151, (2016).

Tým

Mgr. Aleš Benda, Ph.D.

Mgr.
Aleš Benda, Ph.D.

Vedoucí servisní laboratoře, optická (fluorescenční) mikroskopie

ales.benda@natur.cuni.cz
+420325873940
Mgr. Marie Olšinová

Mgr.
Marie Olšinová

Junior Scientist, Advanced fluorescence microscopy

marie.olsinova@natur.cuni.cz
+420325873923
Ing. Petra Prokšová-Grznárová, Ph.D.

Ing.
Petra Prokšová-Grznárová, Ph.D.

Senior Scientist, Live cell microscopy and Flow cytometry

petra.grznarova@natur.cuni.cz
+420325873923
Mgr. Adam Schröfel, Ph.D.

Mgr.
Adam Schröfel, Ph.D.

Senior scientist, Electron microscopy – external member

adam.schrofel@natur.cuni.cz
+420325873924
Mgr. Markéta Dalecká

Mgr.
Markéta Dalecká

Junior scientist, Flow cytometry and Electron microscopy

daleckama@natur.cuni.cz
+420325873923
Galina Kislik

Galina Kislik

Junior scientist, Flow cytometry

galina.kislik@natur.cuni.cz
+420325873940
RNDr. Lenka Koptašiková

RNDr.
Lenka Koptašiková

Junior scientist, Electron and fluorescence microscopy

lenka.koptasikova@natur.cuni.cz
+420325873923
RNDr. Radek Macháň, Ph.D.

RNDr.
Radek Macháň, Ph.D.

Senior scientist, Advanced fluorescence microscopy and data analysis

radek.machan@natur.cuni.cz
+420325873940
Ján Sabó

Ján Sabó

Student, Fluorescence microscopy

jan.sabo@natur.cuni.cz
+420325873924