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Nano-Bio-Imaging Core Facility

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Equipments

3D-SMD

HILO mikroszkóp

3-dimensional single molecule detection (3D-SMD) can be performed using the Highly Inclined and Laminated Optical  (HILO) method using a modified illumination method of Total Internal Reflection Fluorescence Microscopy. Compared to TIRF the angle of incidence is slightly reduced thus the excitation beam does not suffer total reflection but penetrates the sample and illuminates a thin slice of pattern providing a contrasting image in deeper sample planes. The optical axis movement of the sample is performed by a piezo Z-stage thus allowing 3-dimensional imaging of the sample.

Microscope: Olympus IX83
Lasers: 488 nm, 639 nm
Objectives: UMPLFLNW 20x, UAPON OTIRF 100x OIL,
Camera: Hammamatsu Orca-Flash 4.0
Z-stage: Physik Instrumente, PI E-709 piezo Z-stage



Fiber-TIRF

In a Total Internal Reflection Fluorescence Microscope (TIRFM), the excitation beam suffers total reflection at the cover plate-sample interface generating evanescent wave that penetrates into the environment of the cover. The penetration depth of the evanescent wave is 50–100 nm axially into the cell. Accordingly, the evanescent wave makes the fluorescent markers fluoresce in this relatively thin layer. Importantly, TIRFM provides a good signal/noise ratio with good resolution in membrane region. Accordingly, the TIRFM is excellent tool for single molecule tracking.

Microscope: Olympus IX81S1F-ZDC2
Camera: Hamamatsu C9300
Lasers: 491nm, 640nm
Objectives: 10x, 20x, 60x, 100x

TIRF mikroszkóp


SIM

Sim mikroszkóp

In Structured Illumination Microscopy (SIM) an increase in resolution is achieved by varying the excitation intensity as a function of location by means of different illumination patterns. In practice, the excitation beam pattern illuminates the sample from different directions and in different phases. Fourier analysis of the two-dimensional images measured in this way can significantly increases the resolution in the x-y plane. With this imaging method 33 fps can be achieved, that enables the system for detection of dynamics. The disadvantage of this method is the extreme sensitivity to the quality of the illuminating structure. The microscope is available in nano-bio-imaging core facility, is also suitable for living-cell studies as it is equipped with a CO2 and temperature incubator.

Microscope: Zeiss Elyra S.1 
Objectives: Plan-Neofluar 10x/0.30, Plan-APO 40x/1.4 Oil, Plan-Apochromat 63x/1.40 Oil, Alpha Plan-APO 100x/1.46 Oil
Lasers: 405 nm, 488 nm, 561 nm és 642nm
Filters: 405nm excitation (MBS 405 + EF BP 420-480 / LP 750), 488nm excitation (MBS 488 + EF BP 495-550 / LP 750), 561nm excitation (MBS 561 + EF BP 570-620 / LP 750), 642nm excitation (MBS 642 + EF LP 655)
Camera: Andor iXon EMCCD



STED

In the structure of the Stimulated Emission Depletion (STED) microscope  the illumination is supplemented by a so-called STED beam. This STED beam is concentric with the excitation beam with a donut-like intensity profile. The STED beam resets the excited fluorophores by forced emission, depending on its intensity, thus reducing the spot size where effective excitation is achieved. This method provides 50 nm resolution in x-y plane.

System: Abberiror Intruments expert line / FLIM,
Frame: Nikon Eclipse Ti2
Excitation wavelengths: 488 nm, 561 nm, 640 nm 
STED laser: 775 nm
Objective: SR HP Apo F 100xAC OIL
Detector: HybridGasp, APD



STORM

Nikon STORM microscope

We can fit a cylindrically symmetric (Gaussian curve) function on a two-dimensional image of a unique point light source. The centre of point provides the position of the source with high accuracy. However, fluorophores are present at high densities in the labelled samples, so this method is not applicable directly. One possible solution for this problem is the temporal separation of the emission of the fluorophores, that can be achieved by the Stochastic Optical Reconstruction Microscopy (STORM). We only switch on the small portion of originally dark fluorophores before each imaging so the function fitting can be performed without distortion. The activation - imaging steps are then repeated. The repeated steps provide a super-resolution image of the sample.

Microscope: Nikon Eclipse Ti2-E
Lasers: 405 nm, 488 nm, 561 nm, 647 nm
Objectives: Plan Apo lambda 4x, Plan Apo lambda 10x, Plan Apo VC 20x, Plan Apo lambda 60x OIL, SR HP Apo TIRF 100xAC OIL
Confocal detector: Nikon C2
Camera: Hammamatsu Orca-Flash 4.0



In vivo two-photon microscope

The two-photon effect is a process in which a dye molecule is excited by absorbing two photons simultaneously. The cross section of such an event is usually very small, but its probability is proportional to the square of the photon density. In the two-photon microscope available in our core facility, at the focal point of the infrared laser pulse focused on the sample. The light intensity is generated so that the absorbing fluorescent dye in the visible range can be excited by the two-photon effect in a small volume around the focal point (~ 1 um3). Two-photon excitation does not affect parts of the sample outside the focal volume, thus reducing the cell-damage effects of laser beams that limit in vivo studies. Advantages: it can measure up to 800-1200 µm depth, focal excitation, large detection angle, tunable excitation laser wavelength, kHz sampling rate and three-dimensional images can be taken with a scanning method. This configuration is available in the Nano-Bio-Imaging Core Facility providing a platform for studying of brain activity in awake mice.

Microscope: Femto2D-SMART 2-photon microscope
Lasers: Mai Tai HP (690-1040 nm)

In vivo kétfoton mikroszkóp


In vitro two-photon microscope (SRS microscopy)

Stimulated Raman Scattering (SRS) can occur when two photons are absorbed simultaneously. In contrast, to the spontaneous Raman scattering the examined molecule is excited by the simultaneous absorption of the pumping and the Stokes photon. Due to the resonant nature of the process, SRS is more sensitive than spontaneous Raman scattering, however, the system also has the advantage of a two-photon effect. In particular, the sample is excited only in a small volume around the focal point and up to a few mm penetration depth can be achieved. The system is capable of Ca2+ imaging on cells and acute brain slices as well  as label free cellular identification.  

Microscope: Femto3D-AO microscope
Laser: Avesta Topol 1050-C (770-970 nm)



Prof. Dr. Ábrahám István
professor
istvan.abraham@aok.pte.hu
35095
Dr. Makkai Géza
research fellow
makkai.geza@pte.hu
Dr. Jánosi Tibor Zoltán
research fellow
janosi.tibor@pte.hu
Dr. Feller Diána
research fellow
feller.diana@pte.hu
31746

2018

Rapid non-classical effects of steroids on the membrane receptor dynamics and downstream signaling in neurons.

Barabás K, Godó S, Lengyel F, Ernszt D, Pál J, Ábrahám IM, Horm Behav. 2018 Aug;104:183-191. doi: 10.1016/j.yhbeh.2018.05.008. Epub 2018 May 19.

CONTACT
Dr. István Ábrahám
Core Facility Leader
 
 
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