FAQ's about microscopy

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• What are the internal magnifications of the Biorad 1024 and the Biorad Radiance? Answer.
• What's a bandlimited system? Answer.
• How far above and below the in focus information should I sample? Answer.
• What are the pros and cons of piezo-electric vs step motor focusing control? Answer.
• Do I need to use all three channels when observing through a broad bandpass barrier filter? Answer.
• Literature reference on widefield and confocal Nyquist rates? Answer.
• How close may beads be to the image edge before they are rejected? Answer.
• What do you mean by the mounting medium? Answer.
• Which pinhole size to use in case of mechanical hysteresis? Answer.
• May I clip bright pixels which dominate the scene? Answer.
• Is the sampling rate a function of the structure that you're modeling? Answer.
• Is the SNR limited to 16 in 8-bit photon counting? Answer.
• What is an undersampled stack of images? Answer.
• Can we use very small beads in combination with averaging to obtain a PSF? Answer.
• Nyquist rate - What's arcsin/asin? Answer.
• Can artefacts due to unstable laser power be removed by deconvolution? Answer.
• Averaging beads - image size criteria is to small. Answer.
• How to compute the back projected pinhole radius? Answer.
• Backprojected pinhole radius for Biorad confocals? Answer.
• The pinhole size of a measured PSF seems to have no effect on the the restored images. Why? Answer.
• What does the message: 'total reflection at glass/medium interface (...)' mean? Answer.
• Can confocal reflection images be deconvolved? If so, how? Answer.
• What does 'stepsize' mean? Answer.
• How much undersampling is still ok? Answer.
• Is there a suitable source of multi-wavelength micro-beads for PSF measurements? Answer.
• I have heard that imaging diatoms is a difficult task, why? Answer.
• Should PSFs be produced for different wavelength when working in the visible light range? Answer.
• What emission wavelength to use to represent the detected spectrum? Answer.
• What to use as excitation wavelength: laser line or fluorescence spectrum peak Answer.
• How does Huygens Professional derive a Point Spread Function? Answer.
• Can I stretch intensity values? Answer.
• How to analyze images? Answer.
• Huygens excludes all the beads while trying to reconstruct PSFs from beads taken in widefield mode. What is wrong? Answer.
• How should the acquisition zoom factor be taken into account in the calculation for the backprojected pinhole diameter? Answer.
• How to check the size of a bead as it appears in the image? Answer.
• What is more "dangerous" undersampling or oversampling? Answer.
• What is the refractive index of the embedding medium Vectashield? Answer.
• What is the refractive index of the medium? Answer.
• How serious a problem is undersampling in Z-direction? Answer.
• Photon counting - why do people sometimes question its practicality? Answer.
• How deep down (what Z depth) the Huygens would restore 2-photon data. Answer.
• Any suggestions on setting pinholes for spinning disc systems? Answer.
• Which Huygens optical option is best for the Perkin Elmer Ultra View? Answer.
• What to take for the Z-sampling, the nominal or foreshortened distance? Answer.
• A raw 1 micron bead looks very elongated along the optical axis. Why? Answer.
• Which wavelength do I have to set for multi-photon excitation? Answer.
• How to compute the Nyquist rate for an image? Answer.
• Which pinhole size to use in the Leica SP2 confocal microscope? Answer.
• How to estimate the back projected pinhole value from the Airy Disk size? Answer.
• Does the PSF need to be re-measured when one changes the zoom? Answer.
• When is the RI / NA combination correct? Answer.
• What is the quick and dirty way to estimate the SNR in the image? Answer.
• What is an optimal sampling density in Z? Answer.
• How to calculate the X and Y sample sizes using a CCD camera. Answer.
• Can a small sphere like object deconvolved down to a sphere again? Answer.
• Should I use 175 nm or 260nm diameter beads to measure the PSF? Answer.
• What is the difference in sampling between widefield and confocal microscopes? Answer.
• How do I compute the correct sampling for 2-Photon imaging? Answer.
• How to compute the Nyquist rate with 2-photon excitation? Answer.
• What is the potential drawback of estimating the signal to noise ratio (SNR) to high? Answer.
• What is the potential drawback of estimating the signal to noise ratio (SNR) to low? Answer.
• Is deconvolution on 2D or 2D-time images possible? Answer.
• Is it possible to deconvolve brightfield images? Answer.
• Skipping channels in deconvolving with Huygens Pro? Answer.
• Where can I find more references and support about deconvolution? Answer.
• How do I embed beads in water for PSF measurements? Answer.
• Do you know of any suitable solid mountants with suitable refractive indices? Answer.
• Does the pinhole size affect the Nyquist rate? Answer.
• When deconvolving 2-photon data what microscope type should we choose? Answer.
• How to estimate the signal to noise ratio? Answer.
• What is the maximal voxel size at which Huygens can still do a good job. Answer.
• The PSF looks "banana-shaped" in the x-z or y-z planes. What could be the reason? Answer.
• Can we use 1 micron beads for PSF measurements? Answer.
• How do you measure the Numerical Aperture (NA) from the experimental PSF ? Answer.
• How to immobilize a sample with agarose or gelatin Answer.
• What are the characteristics of the Confocal, Widefield, Multiphoton (and other) modules? Answer.
• Should the sampling density used in PSF measurement be equal to the of the specimen? Answer.
• The better sampled the PSF, the better the deconvolution result? Answer.
• What actual resolution improvement can be expected with Huygens? Answer.