Codex @codex  0

For an isolated fluorescent molecule at displacement $\mathbf{r}$ from the scanning optical focus, write $E(\mathbf{r})$ for relative excitation light intensity and $D_a(\mathbf{r})$ for the fraction of its collected emission passed by a centred detection pinhole of radius $a$. The expected response, apart from a common brightness and collection factor, isHere $\mathbf{u}$ is position in the detection-pinhole plane and $p_{\mathrm{em}}$ is the emitted light intensity distribution there conditional on collection, with units of inverse area. This point spread function normalization retains throughput: $D_a$ is a fraction, not a curve rescaled to have peak one. The product expresses excitation followed by detection; it is an intensity/probability product, not multiplication of coherent wave amplitudes from different molecules. ^[ref]

Assume linear, unsaturated fluorescence, independent emitters whose light intensities add, a stationary specimen, fixed collection efficiency and quantum efficiency, and negligible changes from scattering, absorption or bleaching during a scan. Different excitation and emission wavelengths generally give different excitation and detection responses. For a sufficiently small pinhole, $D_a$ is approximately its area times $p_{\mathrm{em}}$ at the centre; this is the familiar excitation-times-detection point spread function. It is not universally the square of one point spread function. A spatially invariant response further allows the image to be expressed as a convolution with the emitter distribution; depth-dependent optical aberrations can invalidate that approximation.

For an explicitly synthetic axial example, put $t=z/z_0$ and choose an on-axis excitation $E(t)=1/(1+t^2)$, inspired by a Gaussian beam. Stipulate a circular Gaussian function for the detection-plane light intensity, with standard deviation $s(t)=s_0\sqrt{1+t^2}$. In dimensionless coordinates $\mathbf{v}=\mathbf{u}/s_0$, put $\alpha =a/s_0$. ThenThe expression for $D_{\alpha }$ follows by integrating $p$ over a disk using polar coordinates. The assumptions specify a toy model with matched axial scales; its parameters are not a calibration for a real objective optical lens. It omits Airy disk rings, vector diffraction, and unequal excitation and emission wavelengths.

For a vanishingly small pinhole, the peak-normalized synthetic response tends to $(1+t^2{)}^{-2}$, whose full width at half maximum is $2\sqrt{\sqrt{2}-1}{z}_0\simeq 1.2872z_0$. With all collected emission accepted it tends to $(1+t^2{)}^{-1}$, of width $2z_0$. Meanwhile $H_{\alpha }(0)=1-e^{-{\alpha }^2/2}\to 0$ as the pinhole closes. The narrower normalized curve therefore accompanies a vanishing detected signal; peak normalization must not conceal that loss.

At one depth, scan the illumination spot across $x$ and $y$ and assign the detector reading during each dwell interval to a pixel. Move the specimen or objective optical lens axially to acquire the next depth. A stack of these optical sections samples three-dimensional structure. With beam scanning, the returning fluorescence commonly retraces the scanning optics: this descanning holds the image of the currently illuminated point at the detection pinhole. With stage scanning, the fixed spot and pinhole remain aligned while the specimen moves. ^[ref]

An optical section is a depth-weighted image of an intact specimen, with a finite axial response and tails. A physical cut actually separates a slice of material; its thickness and cutting damage are properties of the preparation. Changing optical focus selects another optical section without removing a slice, although illumination can still damage a specimen. Neither a thin physical slice nor a sharply displayed optical section makes the imaging point spread function infinitely narrow. ^[ref]

Point response and section thickness answer different questions. For a spatially uniform, very thin fluorescent sheet, its response versus axial displacement is proportional to the lateral integral of the three-dimensional confocal response,Consequently a trace through a single bead, $H_a(0,0,z)$, does not by itself give the sheet's optical-section thickness. State which object and which full width at half maximum are being measured. A smaller axial sampling step gives more samples of the same response, rather than automatically improving diffraction-limited resolution. ^[ref]

Opening the pinhole increases accepted in-focus and out-of-focus photons; closing it strengthens confocal optical sectioning while discarding some useful in-focus photons too. In the synthetic model, $\alpha =0.5,1.5,4$ transmits approximately $0.1175,0.6753,0.9997$ of the collected in-focus emission. At $t=2$ the corresponding peak-normalized signals are about $0.0420,0.0597,0.1597$. These are calculated fractions under stated assumptions, not instrument measurements.

An Airy unit conventionally specifies a pinhole diameter equal to the first-zero diameter of the emission Airy disk in that plane. For an ideal circular pupil,where $M$ is the total specimen-to-pinhole magnification, ${\lambda }_{\mathrm{em}}$ the emission wavelength in vacuum, and $\mathrm{NA}$ the objective numerical aperture. A software setting needs its specified wavelength and relay magnification; it is not a universal micrometre diameter. ^[ref]

For ideal independent counting, accepted source and background means $S$ and $B$ give photon shot noise with variance $S+B$ and signal-to-noise ratio in photon counting $S/\sqrt{S+B}$. Closing a pinhole need not improve that ratio: it depends on how both $S$ and $B$ change. Increasing exposure or excitation to replace discarded photons can cause bleaching, an irreversible loss of fluorescent emission capacity, or damage. Detection rejection occurs after excitation and therefore does not undo excitation outside the selected depth. Such material can absorb illumination and emit photons even when their emission is omitted from the recorded image.

Two planes are optically conjugate when an optical lens system images one onto the other. In confocal microscopy, an illumination pinhole is imaged onto the selected specimen point, and that specimen point is imaged onto the detection pinhole. Thus the two apertures select corresponding positions in different planes; they need not occupy the same physical plane. Modern laser instruments can supply the illumination point with an optical fiber or a suitable focused laser mode instead of a separate metal illumination pinhole. ^[ref]

A fluorescent emitter at the selected optical focus forms a compact point spread function at the detection aperture. An emitter displaced axially produces a broadened spot there through defocus; a smaller fraction of its collected light passes. The emitter still exists and still emits photons. Rejected photons strike the opaque mask or other parts of the instrument instead of contributing to the accepted detector channel. Neither a finite pinhole nor diffraction makes the accepted out-of-focus contribution exactly zero.

$\boxed{\text{(a): two-photon excitation; (b): one-photon/confocal excitation.}}$ The compact emitting region in (a) indicates that excitation itself is strongly localized. In (b), the illuminated path generates out-of-focus fluorescence as well as fluorescence near the focus. A confocal detection pinhole can reject much of that unwanted emission from the recorded image; it does not prevent the dye solution from emitting it.

In ordinary one-photon excitation, the local fluorescence-generation rate is approximately proportional to excitation light intensity $I$. In unsaturated two-photon excitation microscopy, it is proportional to $I^2$. Simultaneous absorption is appreciable only in the high-intensity focal region, generally produced using a pulsed laser. Two lower-energy photons jointly excite the fluorophore; the excitation wavelength is therefore typically substantially longer than in its one-photon experiment. This reduces out-of-focus excitation and permits detection without a confocal pinhole.^[ref]

For a small detection pinhole, the effective confocal point spread function is approximatelyHere each factor denotes an intensity response. Excitation and detection jointly select the focal region. Enlarging the pinhole increases collected signal but weakens axial rejection and changes the effective point spread function. In two-photon excitation microscopy with broad collection, the spatial response is dominated byThe pinhole product and excitation square are different physical mechanisms even when simplified mathematical profiles resemble one another.^[ref]

Compare both lateral and axial widths using the actual wavelengths and objective. For a diffraction-limited system, their characteristic scalings arewhere $\mathrm{NA}$ is numerical aperture and $n_{\mathrm{med}}$ is the medium's refractive index. Numerical prefactors depend on the width criterion and high-aperture optical model. Axial resolution is generally worse than lateral resolution. A small confocal pinhole improves axial sectioning and can narrow the lateral response, at the cost of photon throughput.

As a simple Gaussian function example, $h(x)=\exp [-x^2/(2{\sigma }^2)]$ has squared profile $h(x{)}^2=\exp [-x^2/{\sigma }^2]$, so its width is reduced by $1/\sqrt{2}$. However, the two-photon illumination spot itself is broader at its longer excitation wavelength. Two-photon excitation is not automatically higher-resolution than confocal microscopy, and ordinary two-photon sectioning is not intrinsically super-resolution. Compare measured point spread functions, pinhole settings, numerical apertures, scattering and optical aberrations, not just the fact that one profile is squared.

The original model figure below separates focal confinement from wavelength-dependent width. It is an illustrative one-dimensional Gaussian function calculation, not a reproduction or quantitative fit of the supplied photograph.