DE102006015272A1 - Spectral filter set for LED-based microscope illumination, has excitation filter utilized with higher transmission and steep edge in transmission area, and with smaller optical density in restricted area, by adjusting illumination - Google Patents

Abstract

The set has an excitation filter (1) arranged in an illumination beam path of a microscope. A beam splitter (2) couples an excitation light into an image beam path. An emission filter (3) is arranged in the image beam path, with which the beam splitter is designed with a transmission area. The excitation filter is used with a higher transmission and a steep edge in the transmission area, and with smaller requirements at an optical density in a restricted area, by adjusting LED-BASED microscope illumination.

Description

The The present invention relates to a spectral filter set for LED-based Microscope illuminators, in particular for the effective performance of fluorescence investigations.

To The prior art is known Fluoreszenzeinrichtungen at light microscopes from a light source, which is usually a multispectral, continuous spectrum (xenon lamps - XBO) or a line spectrum (high pressure mercury lamps - HBO) higher intensity emitted, a Auflichtstrahlengang with excitation filter, a beam splitter, for coupling the excitation light in the imaging beam path and an emission filter in the imaging beam path. The passage and stop bands of excitation filter, beam splitter and emission filter are designed so that with the selected spectral range of the light source one possible efficient excitation and detection of one or more fluorophores takes place.

The have in the illumination beam path positioned excitation filter one possible high transmission for the wavelengths, which excite the fluorescence, in particular wavelength radiation retained becomes. The blocking filters positioned in the imaging beam path have however, a high transmission for the lower energy, longer wavelengths of the Fluorescent light, wherein in particular the high-energy excitation light possible Completely is eliminated. The filters used therefore have the task of the desired wavelengths full let through and the undesirable preferably Completely to lock. The difficulty here is that the intensities of Excitation light and emission light is very different. While that Excitation light of high intensity from must be kept away from the microscopic image, the low-emission emission light preferably arrive completely.

To have to the excitation filters all other than the desired wavelength range suppress efficiently, which requires a very high optical density (OD> 5) in the stopband. same for for the Blocking range of beam splitter and emission filter. simultaneously have to the filters as steep as possible Flanks have, so the wavelength ranges of excitation and emission can be used optimally.

filter which meets these requirements in terms of optical density and meet the edge steepness in their preparation very expensive and expensive. On the other hand leads however too low an optical density for so-called "strike-through" of unwanted wavelength to the sample and the detector. Too low a slope in the case of spectrally closely adjacent filters, overlapping in the passband, the so-called "cross-talk" lead, which it to crosstalk Exciting light enters the band of emission light. The fluorescence image loses contrast and by false light artefacts in the picture.

Around one possible Although it is possible to obtain a contrast-rich fluorescence image Intensity of Increase illumination light source, however leads this also to an increase the intensity in the blocked areas and thus to a deterioration of the signal-to-noise ratio in the picture and to a limitation in the exposure time.

One possible High-contrast fluorescence image is obtained when in the microscope used lenses, filters and immersion liquids on the one hand no Have autofluorescence and the other for Fluorescence suitable lenses down to the near UV range have high transmission.

There those in the preparation excited fluorescence is emitted in all directions, it is especially important, possible much of it "to collect". These are lenses used with a large opening angle. With a doubled aperture of the lens can be about four times more Capture fluorescent light. Through the additional use of immersion liquids, especially oil can be additional Eliminate light losses caused by light reflections on the surfaces.

To the prior art are already LED-based lighting units known for microscopes, in which a wavelength selection by dichroic beam splitters and / or filters.

That's how it describes US 6,154,282 A an LED-based illumination unit for fluorescence and phosphorescence microscopes, in which the light emitted by the LEDs is coupled into the beam path via dichroic beam splitters. Furthermore, the use of filters for the selection of the excitation and emission wavelengths is also provided here. By appropriate selection of the illumination source capable of emitting an excitation light within a preselected wavelength band, it can be adapted to the properties of the fluorescent or phosphorescent dye in the sample.

With this solution, an exposure unit for exciting fluorescence and / or phosphorescence is provided, which has a long life and is relatively inexpensive. In addition, an intensity modulation of the exposure source without mechanical diaphragms and / or optical filters possible.

In the US 4,852,985 A1 a lighting unit for microscopes is described in which a plurality of LED's or LED arrays are used as the illumination source. The coupling of the light emitted by the monochrome LED light of three different wavelengths in the optical beam path via beam splitters, wherein the different colored light components are coupled one above the other. In this case, all LEDs emitting the same color are grouped together and arranged in a plane conjugate to a condenser lens of the microscope, concentric with the optical axis of the illumination device.

With the solution described here an exposure unit is provided with which different Illumination types, such as bright field, darkfield and oblique illumination as well as annular Illuminations for microscopic applications can be realized in a simple manner.

In the in the JP 7333516 A described fluorescence microscope, a sample of a plurality of light sources that emit excitation light separated by a dichroic mirror wavelength wavelengths illuminated. The fluorescent light emanating from the sample is likewise separated and detected by wavelength. Due to the wavelength division of the fluorescence light, with simultaneous filtering out of the excitation light, fluorescence images of high brightness can be generated.

Of the The present invention is based on the object, the properties adapt the filter to the light source or the emission light such that by a high edge steepness and transmission in the passage areas and a sufficient optical density in the stop regions fluorescence images produced with high contrast and improved signal-to-noise ratio can be.

According to the invention Problem solved by the features of the independent claims. preferred Further developments and embodiments are the subject of the dependent claims.

Even though the proposed solution a spectral filter set for LED-based microscope illumination, especially for microscopes to carry out of fluorescence studies, a use would be there as well makes sense where the light of an already narrowband illumination source additionally is to be limited and / or disturbing light effects by extraneous light suppress.

The Invention will be described below with reference to embodiments. Show this

1 : the graph of transmission over the wavelength for a spectral filter set in multispectral illumination,

2 : the graph of transmission over wavelength for a spectral filter set in LED based lighting,

3 : an inverse fluorescence image and the gray value curve for a multispectral illuminated sample and

4 : an inverse fluorescence image and the gray value curve for a narrow-band illuminated sample.

at the inventive, from a excitation filter arranged in the illumination beam path of the microscope, a beam splitter for coupling the excitation light in the imaging beam path and an emission filter arranged in the imaging beam path existing spectral filter set for LED-based microscope illuminators for performing fluorescence studies, is the excitation filter as a bandpass filter, the beam splitter spectrally selective, with a transmission range outside the bandpass range of the excitation filter and the emission filter with one, the emission spectrum corresponding transmission area formed. It can in particular by adapting to the LED-based microscope illumination excitation filter with a higher transmission and a steeper flank in the passband, as well as lower Requirements for the optical density OD in the stopband and emission filter with a high optical density OD in the area of the illumination radiation be used.

From The LED-based microscope illumination will provide light with a bandwidth emitted from about 10nm to 100nm, preferably 30-50nm. Out of this light With already very narrow bandwidth is using a as a bandpass filter filtered excitation filter unwanted residual light filtered out, so that the light band of the microscope illumination only one broad from about 20-40 nm.

These very narrow-band illumination radiation is arranged over the in the imaging beam path Spectrally selective beam splitter coupled into these and on the passed to be examined sample. The spectrally selective beam splitter has a transmission range outside the bandpass range of the excitation filter.

In the sample is served by the serving as excitation radiation narrow-band illumination radiation triggered fluorescence. This low-intensity fluorescence radiation is imaged over the transmission range of the spectrally selective beam splitter onto an image output unit, which may be a detector and / or an eyepiece.

From However, the sample is not only emitted fluorescence radiation, but also reflects illumination radiation. The reflected Illumination radiation is to be eliminated from the imaging beam path to obtain a pure fluorescence image on the image output unit. For this purpose, an emission filter is arranged in the imaging beam path, whose Sperrbereich is matched to the illumination radiation and its optical density OD in the entire range of illumination radiation is high.

In addition shows 1 the graph of transmission over the wavelength for a spectral filter set in multispectral illumination. In this case, the microscope illumination emits a multispectral illumination in the form of a continuous or line spectrum. Since in this case a continuous or line spectrum of the same or similar intensity is emitted simultaneously, all unwanted wavelengths must also be suppressed here by an excitation filter. However, the requirements for its stopband due to the suppressed, high-intensity wavelengths are quite high.

There from an excitation filter all other than the desired wavelength range efficiently suppressed is to be in the stopband is a very high optical Density of OD> 5 necessary. The same applies to the beam splitter and the emission filter.

In 1 is the graphical progression of the transmission for the excitation filter 1 , the beam splitter 2 and the emission filter 3 shown in multispectral illumination. The multispectral light emitted by the illumination source is through the excitation filter 1 on the wavelengths of the transmission range 4 limited. For the excitation filter 1 Accordingly, there are high demands on the optical density OD in the stop bands 5 and 6 below and above the transmission range 4 , By representing a spectral range of 300-750nm, the required high slew rate for the excitation filter 1 be highlighted. The narrower this area is, the steeper the edge steepness of the filter must be.

The very complex layered structure of one for the excitation filter 1 , the beam splitter 2 and also the emission filter 3 For multispectral illumination, light transmissions may occur outside the defined transmission ranges. In the transmission curve of the beam splitter 2 shows 1 to an undefined passband 7 that can not be avoided with under.

2 on the other hand, shows the graph of transmission over wavelength for a spectral filter set in LED-based lighting.

in this connection becomes a narrow-band of the LED-based microscope illumination Lighting spectrum (440-540nm) emitted with a bandwidth of 100nm. From that as a bandpass filter With a bandwidth of about 40nm designed excitation filter is only the unwanted Low light filtered out. By adapting to the LED-based microscope illumination the excitation filter has a higher one Transmission and a steeper edge in the passband, as well lower requirements for the optical density OD in the stopband

Also Here is this very narrow band illumination over the arranged in the imaging beam path spectrally selective beam splitter in This coupled and directed to the sample to be examined. The spectrally selective beam splitter has a transmission region for this purpose outside the Bandpassbereiches the excitation filter on.

From the sample is emitted fluorescence radiation and illumination radiation reflected. To the reflected illumination radiation from the imaging beam path To eliminate an emission filter is arranged here, the Sperrbereich is matched to the illumination radiation and its optical density OD in the area of illumination radiation is high, however, may be less in the other restricted areas.

The thus selected fluorescence radiation of low intensity is over the Transmission range of the spectrally selective beam splitter on a Image output unit, which may be a detector and / or an eyepiece, displayed.

The graphic representation according to 2 shows the course of the transmission for the excitation filter 1' , the beam splitter 2 ' and the emission filter 3 ' with narrowband LED-based lighting. The already emitted by the illumination source, already narrowband light is through the excitation filter 1' only slightly on the wavelengths of the transmission range 4 ' limited. To clarify this is also the radiated by the LED-based illumination source spectrum 8th' shown. For the excitation filter 1' exist here only in the restricted areas 9 ' and 10 ' below and above the transmission range 4 ' accordingly high demands on the optical Density OD. In the remaining restricted areas 5 ' and 6 ' the requirements for optical density OD are much lower. Due to the much lower demands on the optical density OD in the stop bands, the edge steepness can be increased and at the same time the complex layered structure can be simplified.

To illustrate the advantages of the proposed solution shows the 3a to examine the fluorescence image of a sample and 3b the associated course of gray values along the in 3a shown diagonal line from top left to bottom right. In the present case, the sample was irradiated with a multispectral mercury high pressure lamp (HBO). A standard spectral filter set was used to block unwanted wavelengths.

For the rest, the show 3a and 3b Images of microtuboli from cells stained with a fluorescent dye. Microtubules, on the one hand, are an integral part of the cytoskeleton and thus responsible for the shape and form of the cell, and on the other hand, they are important for intracellular organization, the transport of vesicles, the positioning of organelles, and for chromosome separation during nuclear division.

In comparison, the shows 4a to examine the fluorescence image of the same sample and 4b the associated course of the gray values also along the in 4a represented diagonal line, in which case the sample was irradiated with a narrow-band LED-based microscope illumination. To block the unwanted wavelengths, the spectral filter set according to the invention was used here.

In the 3a and 4a The fluorescence images of the sample to be examined were recorded under otherwise identical conditions and using the same image output unit in the form of a digital camera.

It can be seen that the gray scale range in 4b about 1.5 times as big as the one in 3b , This results in a much clearer fluorescence image, as the background is blacker and structures can be better adapted and distinguished from one another.

With the spectral filter set according to the invention for LED based Microscope lighting is provided a solution produced with the high-contrast fluorescence images with improved signal-to-noise ratio can be.

While at multispectral light sources are suppressed all unwanted wavelengths have to and the one for that necessary requirements for the corresponding filters are very high, can in the proposed technical solution filters with much lower Requirements are used.

From The LED-based microscope illumination can be any spectrum be emitted as a very narrow band, since the LED's individually controlled and are controllable. Through the LED-based microscope illumination Thus, only excitation light of the desired color in the fluorescence beam path coupled and the excitation filter only serves to unwanted Filter out residual light from the LED spectrum. Thereby are the Requirements for the optical density OD of the excitation filter accordingly lower. Because no high-intensity light filtered out by the excitation filter must be, are much longer exposure times of up to 20 seconds possible, without that thermal reactions occur at the excitation filter. Besides that is the false and scattered light component in the fluorescent image is much lower than when using multispectral light sources in conjunction with conventional Filter sets.

by virtue of it became possible for LED based Microscope illuminators in particular to develop excitation filters, the one higher transmission and slope and thus have a much more effective Enable coupling of excitation light, which in turn to contrast and low noise Fluorescence images leads.

The LED filter sets with their lower requirements on the optical density (OD) in the Barrier area are lighter with a high transmission in the passage area and a steeper edge than that of comparable conventional filter sets the Case is because the number of layers is smaller. Therefore, the LED filter sets less expensive and less expensive produced.

Claims (4)

Spectral filter set for LED-based microscope illumination for carrying out fluorescence investigations, consisting of an excitation filter arranged in the illumination beam path of the microscope ( 1 ), a beam splitter ( 2 ) for coupling the excitation light in the imaging beam path and a arranged in the imaging beam path emission filter ( 3 ), in which the excitation filter ( 1 ) as a bandpass filter, the beam splitter ( 2 ) spectrally selective, with a transmission range outside the bandpass range of the excitation filter ( 1 ) and the emission filter ( 3 ) are formed with a, the emission spectrum corresponding transmission range, wherein by adaptation to the LED-based microscope illumination excitation filter ( 1' ) with a higher transmission and a steeper edge in the passband, as well as lower requirements for the optical density OD in the stopband and emission filter ( 3 ' ) with a high optical density OD in the area of the illumination radiation. A spectral filter set according to claim 1, wherein a LED-based microscope illumination is used by the light with a bandwidth of about 10nm to 100nm, preferably 30-50nm emitted becomes. Spectral filter set according to at least one of Claims 1 and 2, in which an excitation filter ( 1' ) is used to filter out unwanted residual light from the light band microscope illumination, wherein the bandpass has a width of about 40 m. Spectral filter set according to at least one of the preceding claims, in which an emission filter ( 3 ' ) whose stopband is tuned to the illumination radiation and whose optical density OD is high in the region of the illumination radiation and lower in the other stop regions.