NANOBIOLOGY: Advanced (fluorescence) microscopy
Principles of (fluorescence) microscopy & electron microscopy – Basics of optical and fluorescence microscopy
1. Introduction
• Optical microscopy in biology?
o Requirements of techniques: technique needs to have: spatial resolution, temporal resolution,
sensitivity towards organics (proteins, DNA), selectivity
o Goal: see architecture, study dynamics of cells
o We do this at several length scales: organism, organ, tissue, cellular, subcellular, molecular level
o Want to look inside cells: transparency of biological samples; non invasive (in situ and in vivo
experiments); simple/minimal sample preparation wanted
o Conclusion: optical microscopy is often easiest & best solution to study biology
o => need different contrast mechanisms: importance of fluorescence – labelling & label-free
• Summary: important features of optical microscopy:
o (right) Spatial and temporal resolution
o (very good) (single molecule) Sensitivity
o Non invasive: in situ (living cells in celculture) and in vivo (organisms) experiments
o Simple / minimal sample preparation
o Optical transparency
o Wide range of fluorescent probes
▪ Organic: improved quality (spectral, stability, functionality,…) = smaller
▪ Genetic expression (GFP,..) = large = nadeel (can alter behavior of protein, structure)
▪ Quantum dots
o Instrumental improvements
▪ Microscopes, lasers, detectors, optics,…
o Development of specialized data analysis
o Decreased popularity of radioactivity
o Ppt: Eucaryotic – prokaryotic -> µm -> nm
▪ The organism => The organ, a functional grouping of tissues => The tissue, a functional
grouping of cells => Cellular level => Subcellular level => Molecular level
• Different relative sizes of structures/organisms (& microscopy)
o Prokaryotic, eukaryotic, viruses have typical sizes (know them: study length scales)
o Microscopy has limitation in amount of detail they can see:
▪ Light microscopy: limited to 100nm in resolution (Now: pushed to 10nm)
▪ Electron microscopy: smaller scales (but has other disadvantages)
o Exam: open question: virus A & this celltype want to study this process, how do we do this (?)
▪ Proposal: label free or label technique, microscopy for this reason etc; know length scales
(micro or nanometer scale etc)
2. Essential components & terminology in optical microscopy
• Exam: come first with most basic technique that still gives a good answer, because with very advanced
techniques you can almost always answer to all questions but not always as good => Need to find trade-off
• Epi- and transmission (difference in how specimen is illuminated & observed)
o Transmitted light or bright-field microscopy
▪ = light gets transmitted through the sample
▪ 1) In the illumination beam paths, the specimen is placed between the light source (which is
directed onto the sample using a condensor lens) and the objective lens
• => This configuration = dia-illumination, transmitted light, or simply bright field
illumination
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, ▪ 2) The illumination light that has traversed the specimen, and the light that is diffracted by
the specimen are collected by the objective lens
▪ Various light microscopic techniques use this type of illumination
▪ Bright field: homogenous bright background of sample, sample should reduce the light
▪ Algemeen: light source => condenser (lens to illuminate sample) => to sample => objective
(lens to observe sample) => to camera/ eyes for observation
o Epi-illumination:
▪ = illumination & detection is coming from the same side (vb both at the top)
▪ 1) In many cases, however, it is advantageous to illuminate the specimen from the side of
observation vb: when looking at the reflection of opaque samples => In that case, some
optical components of illumination and imaging are identical vb: the objective lens (= also
the one used to illuminate; it serves two roles)
▪ 2) The specimen is illuminated through the objective lens by means of a semitransparent or
dichromatic mirror (see later)
• => This configuration is most commonly used in fluorescence microscopy
▪ Fluorescence microscopy:
• Molecule excited with absorbing wavelength => emission at diff wavelength
• Light coming from same side for illumination & detection: should be shift in
wavelength to separate exciting & emitted light
o Trans & epi: can be in same microscopy body; just different way to observe sample
o Objective lens: optical element that gathers light from the object being observed and focuses the
light rays to produce an image
o Condenser: lens that serves to concentrate light from the illumination source on the object
• Upright or inverted
o = related to observation: done from top (upright), done from bottom (inverted)
o Upright microscope
▪ = the traditional microscope configuration = observation done from top side of sample
▪ The sample is situated below the objective lens which serves to image the sample
▪ Illumination can occur from below (transmission mode) or from the top of the sample (epi)
▪ Disadvantage: difficult to manipulate the object during observation since it is ‘squeezed’
below the objective
o Inverted microscope
▪ = observation done from bottom side of sample
▪ specimens are positioned above the objective lens and therefore observed from below;
▪ epi illumation from below or transmission mode with light source and condensor above the
sample
▪ This is a convenient configuration when samples are examined in open chambers containing
liquid media, for example, cell culture flasks or dishes
▪ Advantage: Specimen manipulation during the microscopic examination such as
microinjection can easily be performed
▪ Advantage: better specimen accessibility + inverted microscopes are generally more stable
▪ Toepassing: examine living cells in culture dishes, standard objectives, avoid sealings, better
access to the stage
• Why different microscopy forms? => related to samples studying
o Inverted: easily access, put one sample after and other to observe (vb glass slide)
o Upright: lens needs to move up, move sample, come down again = more of hassel
• Why then not always use inverted microscope?
o Vb for brainprocesses in rats & mice: you want to easily access the brain, so upright = easier
o Conclusion: which forms of microscopy is related to the experiment
o Opm: upright or inverted microscope don’t influence resolution (detail)
• Upright or inverted: differences
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, o Upright & inverted: determined by size of observation
▪ Upright: top of sample observation + objective at top/condenser bottom inverted:
bottom of sample observation + objective at bottom/condenser top
o Inverted: can have epi illumination: but then light also from bottom side
o Upright: can have epi illumination: but then light also from top side
• Most common light sources (*)
o To obtain optimal imaging performance in the light microscope => the specimen must be properly
illuminated => This requires proper selection of wavelength and intensity (=> light source and filter)
and correct alignment and focus (condensor lens or objective for epi) of the light
• Illuminators & their spectra (*)
o Successful imaging requires delivery to the condenser of a focused beam or light that is bright,
evenly distributed, constant in amplitude, and versatile with respect to the range of wavelengths,
convenience, and cost => Alignment and focus of the illuminator are therefore essential and are the
first steps in adjusting the illumination pathway in the microscope
o 2 groups with many different applications:
o 1) Arc lamps with emission of light over wide range of wavelengths; polychromatic
▪ Useful in brightfield M: observe sample with all different colors of light
o Nr of Lamps
▪ The spectra of two principal lamps used in optical microscopy are shown at the left
▪ 1) Mercury arc lamp with bright emission lines at 365, 405, 436, 546, and 579 nm which can
be selected by the use of color filters for selective excitation of fluorescent molecules
• Significant emission occursa in the UV (below 400 nm) so care has to be taken
▪ 2) Xenon arc lamp have a more or less homogeneous intensity throughout the visible range
and hence serve as good white light sources for many transmission microscopies
o Light-emitting diodes (LEDS) used more & more for general lighting applications (replace lamps)
▪ = are semiconductor junctions that emit photons over a relatively narrow 20–50-nm
bandwidth in the presence of an applied current
▪ The light is distinct from the coherent, 1-nm bandwidth light of a laser
▪ Advantages: Are becoming popular in microscopy because they do not produce heat, do not
produce unwanted flanking wavelengths that must be removed by clean-up filters, last
thousands of hours (longer lifetime tov lamps), produce narrowband spectra that remain
true with ageing, and can be turned on and off rapidly
• + LEDS with specific wavelength also, used for fluorescence
▪ Disadvantage: More broad (wavelength?) distribution tov lasers => So in most sensitive
applications of fluorescence M: always move to lasers
• But LEDs sometimes cheaper, easy solution if no lasers are available
o 2) Lasers with single wavelength; monochromatic light
▪ = excellent for fluorescence: fluorescent molecules absorb monochromatic light & emit
different colors (there is a laser for each fluorescent molecule)
o Filters that adjust light intensity and provide a means for further cleaning up of the spectrum i.e.
selection of wavelengths
o The intensity must be controlled using neutral density filters; similarly, colored glass filters and
interference filters are used to isolate specific color bandwidths.
• Filters for color (wavelength) & intensity adjustment of illumination (fluorescence M)
o Algemeen: Selecting & adjusting the lamp for a particular application = important, but the actual
control of the wavelength and intensity of illumination in a microscope requires the use of filters
o 1) Neutral density filters
▪ = aspecific for the color = regulate light intensity (it reduces intensity equally for all different
wavelengths)
▪ = they have a neutral gray color like smoked glass and are usually calibrated in units of
absorbance (remember Lambert-Beer law) or optical density (OD)
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, • where: A = OD = log10(1/T) = - log(I/I0) (can calculate T)
• with T = transmittance = I/I0 (intensity of transmitted light/ incident light)
• vb: Thus, a 0.1 OD neutral density filter gives 79% transmission and blocks 21% of
the incident light; Other manufacturers indicate the transmittance directly
▪ When stacking multiple ND filters, it is convenient to remember that the total optical density
of the stack equals the sum of the individual filter optical densities.
▪ Advantage: with light on sample you damage biological processes, cause photobleaching
(destruction of fluorescent molecule) => so use little light to study processes: regulate it
o 2) Color filters (the exact mechanism of color selection of both filters should not be known)
▪ = are used to isolate specific colors or bands of wavelengths: because light sources often
contain several wavelengths => efficiently transmits only selected range
▪ Edge filters = are classified as being either
• 1) longpass (transmit long wavelengths, block short ones; above a cut off)
• 2) or shortpass (transmit short wavelengths, block long ones; below a cut off)
• 3) whereas bandpass filters transmit a band/range of (central) wavelengths while
blocking wavelengths above and below the specified range of transmission
▪ Optical performance = defined in terms of the efficiency of transmission and blockage (%
transmission), and by the steepness of the cut-on and cut-off boundaries between adjacent
domains of blocked and transmitted wavelengths
▪ Edge filters = are described by referring to the wavelength giving 50% of peak transmission;
bandpass filters = are described by citing the full width at half maximum (FWHM) and
specifying the peak and central transmitted wavelengths
• FWHM = ange of the transmitted band of wavelengths in nanometers = the distance
between the edges of the bandpass peak where the transmission is 50% of its max
▪ Are essential in fluorescence M: even when using lasers, color filters are essential!!
• Dichromatic mirror enabling epi-fluorescence
o Algemeen: Excitation where it absorbs, detection where it emits
o Figuur links
o Dichromatic mirror = separates excitation light from emission light by reflecting certain wavelengths
& transmitting other wavelengths (other color filters also block or transmit wavelengths)
▪ Arrangement of filters in a fluorescence filter cube
o Diagram: shows orientation of filters in a filter cube in an epi-illuminator for an upright microscope
o Chose laser or lamp with more colors that you select during excitation (excitation filter)
o 1) The excitation beam (yellow line) passes through the exciter (excitation filter: choses only
transmission of light from this region to excite) and is reflected by the dichromatic mirror (under 45°)
and directed toward the specimen (green line) => light collected by objective lens to detector
o 2) The return beam of emitted fluorescence wavelengths (red line) passes through the dichromatic
mirror and the emission filter to the eye or camera
o 3) Excitation wavelengths backreflected or scattered at the specimen are again reflected by the
dichromatic mirror back toward the light source
o 4) Excitation wavelengths that manage to pass through the dichromatic mirror are blocked by the
barrier (emission) filter (prevent scattering of excitation light: no excitation light to detector!)
o Function dichromatic mirror
▪ 1) reflect certain wavelengths (often excitation; illumination), transmits the others (often
fluorescence)
▪ 2) use mirrors for different colors: 2 fluorescent molecules with different emission filters:
separate the fluorescence of the two molecules
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