Optical methods expand fluorescence microscopy

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					                                                                                                                                                                              TECHNOLOGY TUTORIAL

Optical methods expand
fluorescence microscopy
Recent technological advances in conjunction with major developments in fluorescent
markers have made fluorescence microscopy an extremely powerful tool. René Hessling
and Thorsten Kues of Carl Zeiss MicroImaging look at the techniques that are allowing
researchers to study the structures within live cells in ever greater detail.
Fluorescence has become the most impor-
tant contrasting method in light micros-
copy in recent years, largely thanks to the
                                                             Eberhard Fuchs & Susanne Bauch, DPZ Göt tingen

growing family of fluorescence proteins
used for in vivo labelling.
   Fluorescence microscopy provides the
spatial, spectral and temporal resolution
for monitoring dynamic processes inside
living cells, intact tissue and even live
animals. This article offers an overview of
the powerful techniques currently used in
light microscopy.
                                                                                                              A wide-field image (left) compared with an optical section (right) acquired with a Carl Zeiss ApoTome.
Entering 3D-space
In conventional fluorescence microscopy,                                                                      conventional wide-field systems cannot               cules within this limited region, so TIRF
an image consists of light gathered from                                                                      record optical sections directly.                    images do not contain any background
the focal plane as well as from structures                                                                        After image acquisition, software can            fluorescence and have superior contrast
above and below this plane that are not in                                                                    generate optical sections from the wide-             and signal-to-noise ratios. Photobleach-
focus. This is a significant problem when                                                                     field image stack using 3D-deconvolution             ing and phototoxicity are also reduced
studying thick specimens, where light                                                                         (3D-DCV). This process uses informa-                 significantly. As a matter of principle, this
emitted by the out-of-focus regions gener-                                                                    tion from the image formation process to             optical sectioning technique is restricted to
ates a substantial background signal. This                                                                    reassign the out-of-focus signals back to            the coverslip-water boundary and cannot
is because neither the excitation light nor                                                                   the plane from which they originated. In             be used to acquire 3D image stacks. Never-
the emitted fluorescence is restricted to                                                                     contrast to alternative methods described            theless, it can be combined with 3D imag-
the optical axis of the microscope, which                                                                     below, 3D-DCV does not physically dis-               ing on classical wide-field microscopes or
lowers the axial resolution and reduces the                                                                   card any light to generate high-resolution           confocal systems.
image contrast. 3D reconstructions with                                                                       images and is therefore the most sensitive
high spatial resolution are not possible                                                                      method available. When dealing with                  Confocal laser scanning microscopy
from these wide-field images.                                                                                 small and weak fluorescent structures,               In confocal microscopy, optical section-
   The solution to this physically inherent                                                                   3D-DCV is superior to most other optical             ing is achieved by introducing an aperture
problem is optical sectioning. Optical sec-                                                                   sectioning techniques. Its major drawback            diaphragm (pinhole) in a conjugated focal
tions contain only the information from                                                                       is that optical sections are only available          plane of the objective. This pinhole physi-
the focal plane of the microscope and pro-                                                                    after the acquisition of entire 3D stacks and        cally prevents light originating from above
vide images with higher contrast and axial                                                                    elaborate image processing.                          or below the focal plane from reaching the
resolution. Today, several optical section-                                                                                                                        detector. Generally this principle only
ing techniques are available, each with                                                                       Probing the surface with TIRF                        works for a diffraction-limited spot and
their respective strengths and drawbacks,                                                                     Total internal reflection fluorescence               acquiring an image involves scanning the
but all of which have revolutionized micros-                                                                  (TIRF) microscopy is used to investigate             focal point across the sample in both the
copy during the last decade.                                                                                  how molecules interact with surfaces.                x and y directions. These so-called point-
                                                                                                              TIRF involves using an evanescent wave               scanning confocal microscopes provide
3D-deconvolution                                                                                              to create a very thin optical section along          high-resolution and high-contrast optical
Today’s wide-field fluorescence micro-                                                                        the interface between the coverslip and              sections without the need for subsequent
scopes can acquire 3D-image stacks of mul-                                                                    the aqueous medium. The intensity of the             image processing. 3D image stacks can be
tiple fluorescence channels with very high                                                                    evanescent field drops exponentially with            acquired by recording a series of optical
time resolution thanks to fast charge-cou-                                                                    penetration depth and results in an optical          sections at different focus (Z) positions.
pled-device cameras and high-speed LED                                                                        section thickness of around 100 nm. The                Recently, a variation of this technique
sources. However, as mentioned above,                                                                         technique only excites fluorescent mole-             has been developed where an entire line

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                                                                                                                                                                TECHNOLOGY TUTORIAL
                                                                                                    beam is scanned over the specimen in the           stantially. Fully motorized infrared lasers
                                                                                                    object plane and fluorescence only occurs          can be incorporated into confocal laser-
                                                                                                    if at least two photons are absorbed simul-        scanning microscopes, adding the bene-
                                                                                                    taneously by a fluorochrome.                       fits of multiphoton imaging to confocal
                                                                                                       The probability of excitation depends           systems while maintaining ease of use.
Annet te Boehmer, University of Jena

                                                                                                    exponentially on the intensity of the exci-
                                                                                                    tation light, which is at a maximum at the         Experience fluorescence colours
                                                                                                    objective focus. As a result, multiphoton          Today, almost every protein can be tagged
                                                                                                    excitation generates an intrinsic optical          with fluorescent markers and visualized
                                                                                                    section. As in TIRF microscopy, there is no        using fluorescence microscopy. One draw-
                                                                                                     OLESepAdLaserComp 17/12/07 which
                                                                                                    out-of-focus fluorescence excitation, 09:48        back 1
                                                                                                                                                       Page is that the spectral properties of the
                                                                                                    reduces phototoxicity and bleaching sub-           available dyes can limit the experimental
                                       Distinguishing the spectrally overlapping signals of
                                       GFP and YFP-SHP1 fusion proteins in live HEK cells.
                                                                                                                              LASER OPTICS                             IR OPTICS
                                                                                                                              UV-NIR, Nd: YAG                        ZnSe, ZnS, CO2
                                                                                                                             UV Grade High Power
                                       is illuminated and detected. To acquire an                                              Gaussian Mirrors
                                                                                                                                                                    Ge, Si, CaF2, BaF2
                                       optical section, it is only necessary to scan                                           Internet Stocklist
                                                                                                                                                                    Diffractive Optical
                                       the excitation line along the Y axis. This                                                                                        Elements
                                                                                                                                                                      Gratings and
                                       increases the scan speed dramatically and                                                                                     Monochromators

                                       frame rates can reach up to 120 images
                                       a second at 512 × 512 pixel resolution.
                                       High-speed optical sectioning with line-
                                       scanning confocal systems is especially                                                FIBRE OPTICS                              OPTICAL
                                                                                                                               Fibre Patchcords                         FILTERS
                                       beneficial when observing dynamic pro-                                                     Collimators                         UV Filters, NBP
                                                                                                                                Splitters, WDM
                                       cesses in live cells.                                                                   CWDM, Isolators
                                                                                                                                                                        ,     ,
                                                                                                                                                                    LWP SWP Custom
                                                                                                                                                                  Volume Bragg Gratings
                                                                                                                                  Fibre Cable                          Fluorescence
                                                                                                                               Sapphire Cable
                                       Structured illumination                                                                                                        Colour Imaging
                                                                                                                                                                   Raman Spectroscopy
                                       Another method for creating optical sec-
                                       tions for high-resolution multichannel
                                       imaging is structured illumination. Here,
                                       the field stop in the excitation beam path                                             DETECTORS IR                           DETECTORS
                                       of a conventional fluorescence microscope                                                 PbS/PbSe, MCT                               UV-vis
                                                                                                                             Thermopile Pyroelectric                UV SiC with Integral
                                       is replaced by a grid structure, which is                                              Single Chip or Array                     Filters and TIAs
                                       projected into the focal plane of the objec-                                            with or without TEC                   vis Si, NIR InGaAs
                                                                                                                                and/or Integrated                      Colour Sensors
                                       tive. Three raw images are acquired with                                                      Amplifier                        Position Sensing
                                       the grid structure in different positions and
                                       are subsequently combined in real-time to
                                       one optical section.
                                          The principle of structured illumination
                                       is implemented as a simple insert for the                                              LASER DIODES                             OPTO-
                                                                                                                                  Pulsed, CW                        ELECTRONIC
                                       field-stop plane. This can be installed eas-                                                Pigtailed
                                       ily and then upgraded for use in inverted                                                Spectroscopic                      Proximity, Light Barrier
                                                                                                                                                                      Laser Alignment
                                       and upright microscopes.                                                               QCLs, VCSEL, DFB,
                                                                                                                              Lead Salt Optional                           Colour
                                          Structured illumination is particularly                                                TECs, Arrays                        Non-contact Fibre
                                                                                                                               Drivers and Fibre
                                       suited for 2D and 3D multichannel imag-                                                     Coupling
                                       ing of fixed specimen and for slow dynamic
                                       processes within living specimen.
                                       Exploring the depth                                                                          Laser Safety
                                       Generally biological tissues are strongly                                                   IR Converter
                                       scattering materials such that the deeper                                               IR Camera Viewers
                                       the target structure, the greater the absorp-
                                       tion and scattering of visible light. Imaging
                                       structures several hundred microns deep
                                       within tissue at high spatial resolution is
                                       nearly impossible for wide-field and confo-
                                       cal microscopes.
                                          This can be overcome with multipho-
                                       ton microscopy, which uses near infrared                                                                                                   www.lasercomponents.co.uk
                                       laser pulses to excite the fluorescence. Near
                                       infrared light penetrates deeper into tissue
                                       and is generally less phototoxic. Similar to
                                       confocal point scanners, a focused laser

                                       O L E • J a n u a r y 2 0 0 8 • o p t i c s . o r g /o l e
Time Resolved Fluorescence                               TECHNOLOGY TUTORIAL

Combined steady state and
time resolved spectrometer
with microscope accessory and
CCD camera.
o   Time correlated single photon counting
    of fluorescence decays from sub-100ps
    to 50µs
o   Single photon counting for steady state
    and phosphorescence decays from
    10µs to seconds

                                                                                                                                                                                     Carl Zeiss
                                                         The Carl Zeiss ApoTome imaging system produces optical sections using structured illumination.

                                                         freedom. The goal is to distinguish clearly         Beyond imaging
           FLSP920 spectrometer with Microscope
                                                         between the fluorescent markers in order            Different fluorescence microscopy and opti-
                Accessory and Pulsed Laser.              to measure co-localization or functional            cal sectioning techniques allow dynamic
                                                         interaction of the labelled proteins. How-          processes in live cells to be observed. In
    Microscope Accessory                                 ever, this can be difficult even with two           many situations proteins are found in an
                                                         markers due to spectral crosstalk, and the          equilibrium state and are homogeneously
    o   Spectral scanning
                                                         problem grows increasingly complex with             distributed throughout the cell. In this
    o   Fluorescence or phosphorescence time             multiple labels.                                    instance, it is interesting to determine if
        resolved measurements                               One solution to this problem is a pro-           these proteins are stationary because they
                                                         cess known as “emission fingerprinting”,            are bound to the cytoskeleton or if they can
    CCD Camera Accessory                                 in which dyes are distinguished by their            diffuse freely throughout the cytoplasm. In
                                                         spectral characteristics. This method can           order to measure these hidden dynamics,
    o   Sample Imaging
                                                         be applied to wide-field fluorescence micro-        microscopy is used to optically manipulate
                                                         scopes and confocal systems.                        the fluorescent distribution in the sample.
                                                            Certain confocal arrangements acquire               A classical experiment uses the focused
                                                         a “lambda stack” for each image plane,              laser beam in a confocal microscope to
                                                         essentially providing spectral information          bleach the fluorescent signal in a defined
                                                         for each pixel of the image plane. Some             region of the sample and then monitors
                                                         pixels may contain the specific signal from         the diffusion or transport of fluorescently
                                                         just one of the fluorescent dyes in the sam-        labelled proteins back into this region. It is
                                                         ple, while others may contain a composite           possible to measure the dynamic processes
                                                         spectrum, which arises from two or more             by monitoring the speed of fluorescence
                                                         dyes that co-localize in the specimen.              recovery after photobleaching (FRAP).
                                                            Quantitatively separating these dyes is          Multiple variations of this technique such
           Emission Image of Alexa 568 in goblet cells   a three-step process. First, the raw data           as inverse FRAP, fluorescence loss in photo-
                                                         (lambda stack) is recorded. Second, the user        bleaching and fluorescence localization
                                                         specifies which dyes are to be expected in          after photobleaching are routine applica-
                                                         the sample by selecting reference spectra.          tions in confocal microscopy today.
                                                         Finally, a procedure named “linear unmix-              With the development of fluorescent pro-
                                                         ing” determines the relative contributions          teins, such as PA-GFP, KAEDE or DRONPA,
                                                         of the reference spectra to the recorded raw        it is even possible to change the spectral
                                                         data in each pixel and creates images that          characteristics of dyes by turning the fluor-
                                                         display the relative contributions as inten-        escent signals on or off or changing the
        Tel:        +44 (0) 1506 425 300                 sities. The result is a clear and quantitative      colour of the marker by optically manipu-
                                                         separation of fluorescent markers, even             lating the sample.
        Fax:        +44 (0) 1506 425 320 / 326
                                                         those that are spectrally very similar such
        E-mail:     sales@edinst.com                     as Sytox Green and FITC, or GFP and YFP.            René Hessling is head of the training,
                                                         Examples have been published recently               application and support center and Thorsten
        Website: www.edinst.com                          showing the clear separation of up to six           Kues is an imaging and application specialist at
                                                         fluorescent proteins expressed simultane-           Carl Zeiss MicroImaging GmbH. For more
        Address: 2 Bain Square, Kirkton Campus,          ously in live cells.                                information see www.zeiss.de/micro.
                 Livingston, EH54 7DQ, U.K.

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