Modalities and the PSF¶

The modality decides how the optical image is formed from the ground truth. For the modalities available now, that means choosing a point spread function (PSF) and convolving each fluorophore's emission with it.

sim = ms.Simulation(..., modality=ms.Confocal(pinhole_au=1))

Widefield¶

The whole field is illuminated uniformly and imaged onto a camera. Each fluorophore's emission is convolved with the emission PSF. Every plane of the PSF sums to about 1: a defocused point source spreads its light over more pixels, but the total light recorded from it is the same at any focus.

Confocal¶

A focused laser spot is scanned across the sample, and the emission passes a pinhole before reaching a point detector. The confocal PSF is the product of

• the excitation PSF: the relative irradiance of the focused spot, 1 at the center, and
• the effective detection PSF: the emission PSF convolved with the pinhole, which is the fraction of collected light that passes the pinhole for an emitter at each position.

where \(P\) is the pinhole, a disk of diameter pinhole_au, and \(\ast\) is a 2D convolution in each plane. \(\text{PSF}_\text{ex}\) is 1 at the focus, and \(\text{PSF}_\text{em}\) sums to 1 in each plane, so the confocal PSF at the focus is the fraction of the collected light that passes the pinhole.

pinhole_au is the pinhole diameter in Airy units, where 1 AU is 1.22 λ / NA, measured in the sample. Smaller pinholes reject more out-of-focus light and slightly improve resolution, at the cost of signal.

The pinhole has one physical size for all the light that passes it, so λ is one wavelength per pinhole, not the wavelength of each fluorophore:

• By default, λ is the center of the channel's emission filter passband (e.g. 525 nm for a 525/50 filter). As on many point scanners, the channels of a track are detected through one pinhole, and λ is the average of their band centers. With shared_pinhole=False, each channel has its own pinhole, matched to its own band.
• If a channel sharing the pinhole has no band-limited emission filter (e.g. only a longpass), λ is the mean wavelength of the emission detected in all the channels sharing it, and microsim warns.
• airy_unit_wavelength_nm sets λ directly.

The excitation spot is computed at the excitation wavelengths. With several lasers, or a broadband source, the light each fluorophore absorbs is grouped into bands at most excitation_bin_width_nm wide (Settings), and the spot is the sum of the spots at each band's wavelength, weighted by the light absorbed in it.

For a point-scanning modality:

• power on the channel is the peak irradiance at the center of the spot (often kW/cm² or more), not an average over the field.
• exposure_ms is the pixel dwell time (often 0.0005–0.01 ms), and is not divided among pixels. Channels acquired together (in one track) must have the same dwell time.
• Saturation is applied at each position of the excitation spot rather than to the emission rate.
• Downsampling to the output space averages rather than sums (Spaces).
• Point-scanning microscopes record with a point detector (PMT or HyD), but microsim accepts any detector.

Identity¶

Identity applies no PSF. Use it when the ground truth is already a microscope image (for example, a real widefield image) and you are interested in the spectral and detector parts of the simulation: fluorophores, filters, bleedthrough, and noise: modality=ms.Identity(), or {"type": "identity"}.

Objective lens¶

ObjectiveLens sets the numerical aperture and the refractive indices used by the PSF model. The default is a 1.4 NA oil immersion objective.

ms.Simulation(
    ...,
    objective_lens=ms.ObjectiveLens(
        numerical_aperture=1.2,
        immersion_medium_ri=1.333,
        immersion_medium_ri_spec=1.333,
        specimen_ri=1.333,
    ),
)

The *_spec parameters describe the conditions the objective is corrected for. When the actual values differ (a coverslip of the wrong thickness, or the wrong immersion oil), the PSF shows the resulting spherical aberration.

The NA also sets the collection efficiency (the fraction of emitted light that enters the objective), so a lower NA gives a dimmer image as well as a larger PSF.

Emission spectrum and the PSF¶

The PSF depends on wavelength, and a fluorophore emits over a range of wavelengths. By default, microsim computes one PSF per fluorophore and channel, at the centroid wavelength of the detected emission. For a more accurate result, it can split the detected emission into several wavelength bins and sum one PSF per bin, weighted by the photons in that bin:

ms.Settings(spectral_bins_per_emission_channel=3)

This takes proportionally longer. See Settings.

PSF caching¶

PSFs are cached in memory and on disk, keyed by every parameter that affects them, so repeating a simulation with a different sample, or with a detector with the same QE, does not recompute them. (The QE changes the spectrum of the detected light, and so the wavelengths at which the PSFs are computed.) See Settings.