You can expect some 'bleed through', this is a manageable problem. Having determined the level of a background signal, the background settings are adjusted accordingly and it's eliminated from the final image. If you have a good signal to noise ratio to begin with, little effect will be seen in your image, and you can be confident in the results.

Cameralensmount chart

Small Optic AdaptersFor small optics (Ø10 mm or less), we offer glue-in stainless steel adapters, as shown to the right, that allow optics with various diameters ranging from Ø2.0 mm to Ø9.0 mm to be mounted in our SM05-threaded mounts, and optics with a Ø10 mm diameter to be mounted in our LMR18 mount. We also offer a line of small optic adapters with external SM1 threading, which can mount optics from Ø5 mm toØ20 mm in SM1-threaded lens mounts.

These mounting rings feature internal SM05, SM1, or SM2 threading that extends the full length of the mount without any retaining lip. They are perfect for mounting lens tubes and mounted optics, and can also be used to hold unmounted optics when used with two retaining rings (sold separately below).

Lensmount index

For retaining rings not covered by these cylindrical spanner wrenches, please see the SPW801 Adjustable Spanner Wrench sold at the bottom of the page. The SPW801 features an adjustable spanning range as well as long blades that extend 1.6" (40.7 mm) from the body of the wrench. Hence, for lenses with a high radius of curvature, we recommend using the SPW801 over the cylindrical spanner wrenches, whose guide flanges may come into contact with the lens.

Moving the point of focus up and down moves the optical section through the sample, a so called 'z series' because it moves along the z axis.

Metric threads have a slightly more complex tolerancing method that uses tolerancing grades, designated by a number 3 through 9; and tolerancing positions, which use letters e through h. Grades provide a measure of the tolerance itself: the smaller the number, the tighter the tolerance. Positions denote the distance of the tolerance from the pitch diameter. Uppercase positioning letters indicate internal threads while lowercase positioning letters indicate external threads.

As shown below, the SPW602, SPW603L, and SPW608 wrenches feature an engraved scale to indicate spanner wrench depth in both imperial and metric increments (0.02" and 0.5 mm, respectively), thereby enabling precise positioning of optics.

Fixed Lens Mounts with Internal Threads (LMRx Series)Our standard fixed optic mounts (LMRx series) are available for 20 distinct optic diameters ranging from Ø5 mm to Ø4", feature internal threading, and come complete with one retaining ring for securing the optic in place. A bottom-located 8-32 (M4) tapped hole is provided for mounting on Ø1/2" posts. Our most popular fixed optic mounts in this series, the LMR05(/M), LMR1(/M), and LMR2(/M), are available individually or in packs of 5 for convenience. The LMRx series also has vacuum-compatible versions: these unanodized 6061-T6 aluminum mounts are compatible with environments down to 10-6 Torr directly out of the package.

The main thing to decide is which laser(s) you'll need to excite your fluorescent sample. It's important to remember that the confocal is essentially just another form of fluorescence microscope. Before going anywhere near it, check your labelling works by using a conventional fluorescence microscope. If your procedure doesn't work under those conditions, it's unlikely to work on the confocal. Doing this simple test will save you a lot of time – and cost.

For optimal visual alignment of the beam, these plates feature laser-engraved concentric circles, with the diameter of each circle being 2 mm greater than the previous.

CameraLensMount Adapter

The SPB600 Spanner Wrench Bit Kit includes bits for SM05 (0.535"-40), SM1 (1.035"-40), and SM2 (2.035"-40) retaining rings; a driver handle with a 1/4" hex socket; and a carrying case. In addition, the case has a location to house the SM3 (3.035"-40) spanner bit available separately below.

These Fixed Optic Mounts are our standard mounts for 20 distinct optic diameters ranging from Ø5 mm to Ø4". Each mount features internal threading, and ships with a retaining ring for securing the optic into place. The internal threading is deep enough to hold most unmounted optics or externally threaded components. The base of each mount features an 8-32 (M4) tapped hole, which allows for easy mounting to Ø1/2" Posts, as shown to the right. Additional retaining rings are sold separately below.

Each bit includes guide flanges that prevent most optical elements from being scratched while the bit is being inserted. When using lenses with high curvature, we recommend the SPW801 Adjustable Spanner Wrench sold below, which uses long blades that prevent scratches caused by the guide flanges.

These vacuum-compatible fixed lens mounts are fabricated from unanodized 6061-T6 aluminum and are suitable for vacuum chamber and clean room applications. Each of these fixed lens mounts features a hole in the back which is connected to the 8-32 (M4) tapped mounting hole on the base in order to serve as a vent, allowing trapped gas to escape when a vacuum chamber is pumped down. This vented mounting hole allows for easy integration with our vacuum-compatible Ø1/2" posts. Do not plug this vent hole or attempt to use it as a mounting point. These lens mounts are available for Ø1/2", Ø1", and Ø2" optics and each one is shipped with a vacuum-compatible retaining ring for securing an optic against the retention lip at the back of the mount.

Thorlabs also offers an extensive line of adapters for unmounted optics which allow optics ranging from Ø2 mm to Ø2" to be mounted in our fixed optic mounts.

Our vacuum-compatible lens mounts and included retaining rings are packaged in double airtight bags for cleanroom and vacuum chamber applications.

On the Zeiss 710 the emission detection ranges can be customised to suit the fluorophore you're using. Just adjust the slider to suit.

Lasers are intense, but excite over a very narrow range compared to conventional mercury burner light sources. You should therefore check the excitation curve of your fluorophore to ensure it will be excited enough. Most excitation curves are bell shaped, you don't have to hit the maximum peak, but excitation efficiency drops away as you move down the shoulders of the curve.

Fixed Lens Mounts with Internal and External Threads (LMRxS Series)Thorlabs also offers fixed mounts with both internal and external SM threading (LMRxS series), which are available for optic diameters of Ø1/2", Ø1", and Ø2". Each LMRxS series mount also features an 8-32 (M4) tapped hole for Ø1/2" post mounting and comes complete with a compatible retaining ring.

Thorlabs' Adjustable Spanner Wrench features a spanning range that is continuously adjustable from 0.12" to 2.89" (3.0 mm to 73.4 mm). It is capable of providing an exact fit for retaining rings as large as the SM3RR for Ø3" Lens Tubes and Mounts. The diameter spanned by the blades remains centered while the knurled knob is adjusted.

The Ø1/2" Optic Mounting Adapters featured below are compatible with our LMR05(/M) or MLH05(/M) Lens Mounts, SM05 Lens Tubes, or 16 mm Cage Components. The LMRA10 is compatible with our LMR18(/M) mount, AD18T adapter, or SM1AD18 adapter. Once the optic is secured in the small-optic adapter, the new assembly is held in one of these threaded mounts using an SM05RR or SM18RR Retaining Ring.

Thread SeriesMost screws are identified by their thread series. Thread series are denoted by the major diameter and density of threads. Unified threads specify density in threads per inch, while Metric threads specify the thread pitch. For example, in the Unified nomenclature, a 1/4"-20 cap screw has a 1/4" diameter barrel and the pitch is 20 threads per inch (TPI). In metric nomenclature, an M4 x 0.7 cap screw has a 4 mm barrel and the pitch is 1 thread per 0.7 mm. The term M4 x 0.7 is often shortened to just M4.

Test the inherent fluorescent property of your sample with control slides with no markers. if you are using more than one fluorophore make a slide with each of the markers. These are then examined with the 'wrong' detector, to see if they might contribute any unexpected signal to that channel.

This page also features alignment plates for simple beam alignment, threaded retaining rings, spanner wrenches, and spanner bits for easy optic installation.

Lensmount types

These Mini-Series Fixed Lens Mounts are compact, lightweight mounts for seven distinct optic diameters ranging from Ø5 mm to Ø1/2". Each mount features internal threading and ships with a retaining ring for securing an optic into place. The internal threading is deep enough to hold most unmounted optics or externally threaded components. The base of each mount features an 4-40 (M3) tapped hole, which allows for easy mounting to Ø6 mm Mini-Series posts, as shown to the right. Additional retaining rings are sold separately below.

Three of our most popular fixed optic mounts, the LMR05(/M), LMR1(/M), and LMR2(/M), are available individually and in packs of 5. 5-packs include one retaining ring per mount for a total of five rings.

Features of a ThreadA thread consists of three repeating features: a crest, flank, and root (see drawing to the right). Except in special cases, threads have symmetrical sides inclined at equal angles when a vertical line is drawn through the center of a crest or root. The distance between corresponding points on adjacent threads is known as the pitch of the thread. The flank angle is defined as the angle the flank makes with a perpendicular ray drawn from the screw axis. Unless otherwise stated, threads have a flank angle of 30°, resulting in a total angle between flanks of 60°. Each feature is shown in the diagram to the right.

Quoting from the Machinery's Handbook, 29th Edition, p. 1885: "To designate the tolerance class, the grade and position of the pitch diameter is shown first followed by that for the major diameter in the case of the external thread or that for the minor diameter in the case of the internal thread, thus 4g6g for an external thread and 5H6H for an internal thread. If the two grades and positions are identical, it is not necessary to repeat the symbols, thus 4g, alone, stands for 4g4g and 5H, alone, stands for 5H5H."

Mounted lensnikon

Thread ClassThe tolerances and allowances on a thread series are given by a thread class. Unified thread classes are alphanumeric identifiers starting with a number from 1 through 3, where 1 is the loosest tolerance and 3 is the tightest, and either A for external threading or B for internal threading.

Vacuum CompatibilityOur vacuum-compatible lens mounts are chemically cleaned and prepared for vacuum applications before packaging. They are compatible directly out of the packaging with vacuum environments down to 10-6 Torr. With additional cleaning and processing, they can be used at even lower pressures, only limited by the outgassing rate of the aluminum. The material properties of the aluminum and the cleaning methods completed by the end user should be used to determine the appropriateness of these products and materials in a specific vacuum system.

Due to the large amount of control you have in the confocal software to adjust intensity, background, etc, it's very important to have good control samples. All the signal reaching the detectors is coloured with the appropriate false color, e.g. FITC signal is coloured green. Consequently, any auto-fluorescence or stray signal from another fluorophore will be coloured exactly the same as legitimate signal. These subtle effects can often be seen down the eyepiece of the microscope as shifts in hue, something the confocal will not show you!

Thread FormA thread form is a set of rules that define the features' scale relative to one another. Common thread forms include the Unified Screw Thread Form, used in the United States of America and measured in imperial units, and the ISO Metric Screw Thread Form, used in many parts of the world and measured with the International System of Units. There are many thread forms in the Unified screw thread standard designated by either UN, which defines a flat root contour, or UNR, which defines a round root contour. These can be further described by appending more letters. For example, an extremely fine thread with a flat root contour is designated UNEF. Those forms which are not standardized by the Unified screw thread system are designated UNS.

Mounted lensfor photography

Thorlabs' SM Series ThreadsThreading specifications for our SM threads, utilized in our lens tube and cage system components, are given below so that you can machine mating components to suit your application. Most SM series threads utilize a non-standard Unified thread form, indicated by the letters UNS, with a 30° flank angle and a thread class of 2A and 2B. The exception is our SM30 series thread, which is a Metric thread form with a 30° flank angle and a tolerance of 6H/6g. We also offer products with C-Mount and RMS threads, and the specifications for these threads are given below for reference. Please note that other manufacturers may have different tolerances for C-Mount and RMS threads. For other thread specifications that are not listed here, please contact Tech Support.

The following is a general overview of screw threading. For more details regarding specifications and dimensions, please consult the Machinery's Handbook, available for purchase at many bookstores.

By eliminating everything which is out of focus, the confocal microscope takes an 'optical section' through a sample. Going back to the photography analogy, only the person's face would be visible, the trees in the background and objects in front would be black.

Histologists section material to around 4µm thick, because when it's this thin most of the specimen will lie within the focal plane – and therefore be in focus from top to bottom. However, go to say a 60× objective and you'll notice there is a perceptible depth to the section as regions go in and out of focus. Consequently, to obtain a sharp image under conventional (wide-field) light microscopy anything thicker than about 4µm becomes too blurry to be useful. It's not that there isn't a sharp plane of focus lurking in there, it's just obscured by too much out of focus information. To utilise the 'third dimension' or z axis of a sample then requires either many serial sections – or a confocal.

The major diameter is taken from the crests of a thread while the minor diameter is taken from the roots. For most screws, crests and roots do not terminate at a sharp point, so crest and root truncation values are included in the definitions of major and minor diameter. The pitch diameter is approximately halfway between the major and minor diameters.

Mini-Series Fixed Lens Mounts with Internal Threads (MLHx Series)Our Mini-Series Fixed Optic Mounts (MLHx series) are available for seven distinct optic diameters ranging from Ø5 mm to Ø1/2", feature internal threading, and come complete with one retaining ring for securing the optic in place. A bottom-located 4-40 (M3) tapped hole is provided to mount directly to our Ø6 mm Mini-Series posts or Ø12 mm pedestal posts. Our Mini-Series post holders allow our Ø6 mm posts to be mounted onto our Mini-Series breadboards.

A confocal laser scanning microscope removes out-of-focus blur. Samples need to be either fluorescent or reflective. Most biological samples are naturally fluorescent (auto-fluorescent) or have artificial fluorescence added to specific regions to make them visible.

The reversible blades have a flat tip on one end intended for use with retaining rings, and an edge-offset fine point on the opposing end for adapters with holes. The blades extend 1.6" (40.7 mm) from the body of the wrench. Hence, compared to our cylindrical spanner wrenches sold above, the SPW801 is ideal for securing lenses with a high radius of curvature because the guide flanges on the cylindrical spanner wrenches may come into contact with the lens. To reverse the blades, a 0.050" (1.3 mm) balldriver or hex key is needed. Replacement blade pairs are available separately.

These Retaining Rings are replacements for those that are provided with our fixed lens mounts featured above. Two retaining rings are required for mounting optics in the fixed lens mounts with no retaining lip (item # SMRx). We also offer stress-free and plastic retaining rings for our Ø1.0" lens mounts, and retaining rings in additional sizes on our main Retaining Rings page.

If using these bits with a power drill, care should be taken to avoid overtightening the retaining ring due to the added torque created by the tool. Electric tools should be used at a low speed, and we recommend that the final tightening of the retaining ring be done by hand.

C-MountLens

These spanner wrenches enable adjustment or installation of many of our standard retaining rings. The hollow construction of each spanner wrench allows the retaining ring to be adjusted without obscuring the beam path. They feature knurled handles for improved grip as well as flanges to keep the retaining ring on the end of the spanner wrench while inserting or removing the ring.

The internal threading is deep enough to hold most unmounted optics, while the external threading is deep enough to mount lens tubes, irises, or other internally threaded SM components (see picture to the right). These mounts provide a compact solution to mount multiple SM-threaded components on a single post.

Fixed Lens Mounts with No Retaining Lip (SMRx Series)To mount multiple lens tubes or mounted optics, Thorlabs offers a line of internally SM-threaded mounts with no retaining lip (SMRx series), available for optic diameters of Ø1/2", Ø1", and Ø2". These mounts can also be used to hold unmounted optics when combined with two retaining rings (not included). Like the LMRx and LMRxS series, these mounts also feature an 8-32 (M4) tapped hole for mounting on Ø1/2" posts.

Mounted lenscanon

These alignment targets can be quickly and easily dropped onto one of our fixed lens holders or lens tubes allowing the user to align a visible beam using the center Ø1 mm through hole. Once the beam is aligned, the tool can be lifted from the lens holder or lens tube without disturbing the alignment and can later be replaced into approximately the same position.

For ordering convenience, our Ø1/2" and Ø1" Retaining Rings, the SM05RR and SM1RR, are available in packs of 10, and our Ø2" Retaining Ring, the SM2RR, is available in packs of 5.

These fixed lens mounts, which feature internal SM threading on one side and external SM threading on the other, are available for Ø1/2", Ø1", and Ø2" optics. The mount holds an optic securely with the included retaining ring and can be post mounted to a Ø1/2" Post.

A useful example to understand confocal, is when you photograph a person's head. You might notice the trees in the background are blurred, as are features in front of the person. This is because the person's head lies in the focal plane. Objects in front or behind the focal plane gradually go more and more out of focus. Exactly the same thing happens in a microscope slide.

The control panel of the Zeiss LSM 710 confocal may look intimidating, but you'll probably only need to become familiar with a small proportion of the components.

Generally, if you've successfully stained your samples and viewed the results under an ordinary epi-fluorescence microscope, your samples will be suitable for confocal.

Spanner Bits allow SM-threaded retaining rings to be installed or adjusted without damaging optics that may be in place. They feature a standard 1/4" hex shank that allows them to be used with a manual driver handle, such as the one included in the kit below.

Thorlabs' small-optic mounting adapters are designed to mount non-standard optic sizes into Ø1/2" or Ø18 mm Mounts. Each stainless steel adapter includes two or three reservoirs next to the lens cell to secure the lens in place with General Purpose or UV Curing epoxy. Simply place a drop or two of adhesive into each reservoir located next to the lens in the adapter. We recommend using our NOA61 UV curing adhesive and MS403-10 epoxy syringe.